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Alzheimer’s Disease (AD) is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterised by the presence of amyloid plaques, intracellular neurofibrillary tangles and pronounced cell death. The aim of this thesis was to investigate pathways involved in the Aß cascade of neurodegeneration. Since novel findings indicate that already this Aß species exerts neurotoxic effects long before hyperphosphorylated tau, neurofibrillary tangles and extracellular Aß plaques appear, the investigations were accomplished with specific regard to the effects of intracellular Aß. The Swedish double mutation in the APP gene results in six- to eightfold increased Aß production of both Aß1-40 and Aß1-42 compared to human wildtype APP cells (APPwt). Data obtained from PC12 cells indicate that it is possible to specifically increase the Aß load without enhancing APP expression levels. On the basis of these findings, it seemed possible to investigate dose-dependent effects of Aß in multiple experimental designs. These assay designs were created in order to mimick different in-vivo situations that are discussed to occur in AD patients: APPsw PC12 cells exhibit low physiological concentrations of Aß within picomolar range in contrast to APPsw HEK cells, expressing Aß levels within the nanomolar range. Of note, the APPsw HEK cells showed a specific and highly significant increase in the intracellular accumulation of insoluble Aß1-42. Moreover, an intracellular accumulation of Aß and APP was found in the mitochondria of the HEK APPsw cells suggesting a direct impact on mitochondrial function on these cells. This effect might finally lead to disturbances in the energy metabolism of the cell or to increased cell death. Furthermore, baseline g- and ß-secretase activity was assessed since these enzymes represent promising therapeutic targets to slow or halt the disease process. As expected, ß-secretase activity was significantly elevated in all APPsw cell lines. This might be due to the proximity of the Swedish double mutation next to the N-terminus of the Aß sequence. Interestingly, g-secretase activity was similarly increased in PC12 APPsw cells. In addition, the toxicity of different Aß species was investigated in SY5Y and PC12 cells with regard to their effect on cellular viability mirrored by mitochondrial activity using MTT assay. Here, it turned out that not monomers, but already dimers are neurotoxic correlates. Fibrillar Aß species showed the highest toxicity. In the next step, SY5Y cells forming endogenous, dimeric APP and Aß were investigated. In accordance with previous findings, these cells showed a decreased MTT reduction potential in comparison to APPwt and control SY5Y cells reflecting a decrease of cellular viability. The impaired energy metabolism of the cells was even more drastically mirrored by reduced baseline ATP levels. In the second part of this thesis, the expression and intracellular distribution of Bcl-2 family proteins and pro-apoptotic mitochondrial factors under baseline conditions and during oxidative stress were analyzed in the APPwt and APPsw bearing cells. The most prominent finding was the reduction of expression levels of the anti-apoptotic factor Bcl-xL in the cytosolic fractions of APPwt and APPsw PC12 cells. This might indicate that a lack of anti-apoptotic factors or their altered intracellular distribution, rather than an increase in caspase-dependent pro-apoptotic factors, could be responsible for the increased vulnerability of APPwt- and APPsw-transfected PC12 cells against oxidative stress. Since total Bcl-xL expression was unaffected in PC12 cells, in contrast to APPwt and APPsw-expressing SY5Y and HEK cells revealing significantly decreased Bcl-xL expression levels. Thus, alterations in Bcl-xL distribution seem to be an early event in the disease process. Increasing Bcl-xL expression might potentially be one promising strategy for AD modification. PC12 and HEK cells bearing APPsw or APPwt were treated with the potent g-secretase inhibitor DAPT. Of note, DAPT did not only efficiently block Aß production, but additionally led to an elevation of the MTT reduction potential, reflecting an increase in cellular viability. As another disease-modifying strategy, several efforts are undertaken to ameliorate AD-relevant symptoms by the treatment with nerve growth factor (NGF). Generally, it is known that substituted pyrimidines have modest growth-promoting effects. Here, KP544, a novel substituted pyrimidine, was characterised. This drug increased MTT reduction potential in terminally differentiated and undifferentiated PC12 cells. Furthermore, treatment with KP544 led to a reduction in Aß1-40 secretion. Thus, one may conclude that the target of KP544, GSK-3ß, represents a connecting link between the two main pathological hallmarks of AD and might thus be a very promising therapeutic target for AD.
Cysteinyl leukotriene receptor 1 antagonists (CysLT1RA) are frequently used as add-on medication for the treatment of asthma. Recently, these compounds have shown protective effects in cardiovascular diseases. This prompted us to investigate their influence on soluble epoxide hydrolase (sEH) and peroxisome proliferator activated receptor (PPAR) activities, two targets known to play an important role in CVD and the metabolic syndrome. Montelukast, pranlukast and zafirlukast inhibited human sEH with IC50 values of 1.9, 14.1, and 0.8 μM, respectively. In contrast, only montelukast and zafirlukast activated PPARγ in the reporter gene assay with EC50 values of 1.17 μM (21.9% max. activation) and 2.49 μM (148% max. activation), respectively. PPARα and δ were not affected by any of the compounds. The activation of PPARγ was further investigated in 3T3-L1 adipocytes. Analysis of lipid accumulation, mRNA and protein expression of target genes as well as PPARγ phosphorylation revealed that montelukast was not able to induce adipocyte differentiation. In contrast, zafirlukast triggered moderate lipid accumulation compared to rosiglitazone and upregulated PPARγ target genes. In addition, we found that montelukast and zafirlukast display antagonistic activities concerning recruitment of the PPARγ cofactor CBP upon ligand binding suggesting that both compounds act as PPARγ modulators. In addition, zafirlukast impaired the TNFα triggered phosphorylation of PPARγ2 on serine 273. Thus, zafirlukast is a novel dual sEH/PPARγ modulator representing an excellent starting point for the further development of this compound class.
In the past, the genetically diabetic-obese diabetes/diabetes (db/db) and obese/obese (ob/ob) mouse strains were used to investigate mechanisms of diabetes-impaired wound healing. Here we determined patterns of skin repair in genetically normal C57Bl/6J mice that were fed using a high fat diet (HFD) to induce a diabetes-obesity syndrome. Wound closure was markedly delayed in HFD-fed mice compared to mice which had received a standard chow diet (CD). Impaired wound tissue of HFD mice showed a marked prolongation of wound inflammation. Expression of vascular endothelial growth factor (VEGF) was delayed and associated with the disturbed formation of wound margin epithelia and an impaired angiogenesis in the reduced granulation tissue. Normal wound contraction was retarded and disordered. Wound disorders in obese C57Bl/6J mice were paralleled by a prominent degradation of the inhibitor of NFκB (IκB-α) in the absence of an Akt activation. By contrast to impaired wound conditions in ob/ob mice, late wounds of HFD mice did not develop a chronic inflammatory state and were epithelialized after 11 days of repair. Thus, only genetically obese and diabetic ob/ob mice finally developed chronic wounds and therefore represent a better suited experimental model to investigate diabetes-induced wound healing disorders.
BACKGROUND: Micro-RNAs (miRNA) are attributed to the systems biological role of a regulatory mechanism of the expression of protein coding genes. Research has identified miRNAs dysregulations in several but distinct pathophysiological processes, which hints at distinct systems-biology functions of miRNAs. The present analysis approached the role of miRNAs from a genomics perspective and assessed the biological roles of 2954 genes and 788 human miRNAs, which can be considered to interact, based on empirical evidence and computational predictions of miRNA versus gene interactions.
RESULTS: From a genomics perspective, the biological processes in which the genes that are influenced by miRNAs are involved comprise of six major topics comprising biological regulation, cellular metabolism, information processing, development, gene expression and tissue homeostasis. The usage of this knowledge as a guidance for further research is sketched for two genetically defined functional areas: cell death and gene expression. Results suggest that the latter points to a fundamental role of miRNAs consisting of hyper-regulation of gene expression, i.e., the control of the expression of such genes which control specifically the expression of genes.
CONCLUSIONS: Laboratory research identified contributions of miRNA regulation to several distinct biological processes. The present analysis transferred this knowledge to a systems-biology level. A comprehensible and precise description of the biological processes in which the genes that are influenced by miRNAs are notably involved could be made. This knowledge can be employed to guide future research concerning the biological role of miRNA (dys-) regulations. The analysis also suggests that miRNAs especially control the expression of genes that control the expression of genes.
Background: Oral anticoagulant therapy (OAT) with warfarin is the standard of stroke prevention in patients with atrial fibrillation. Approximately 30% of patients with cardioembolic strokes are on OAT at the time of symptom onset. We investigated whether warfarin exacerbates the risk of thrombolysis-associated hemorrhagic transformation (HT) in a mouse model of ischemic stroke.
Methods: 62 C57BL/6 mice were used for this study. To achieve effective anticoagulation, warfarin was administered orally. We performed right middle cerebral artery occlusion (MCAO) for 3 h and assessed functional deficit and HT blood volume after 24 h.
Results: In non-anticoagulated mice, treatment with rt-PA (10 mg/kg i.v.) after 3 h MCAO led to a 5-fold higher degree of HT compared to vehicle-treated controls (4.0±0.5 µl vs. 0.8±0.1, p<0.001). Mice on warfarin revealed larger amounts of HT after rt-PA treatment in comparison to non-anticoagulated mice (9.2±3.2 µl vs. 2.8±1.0, p<0.05). The rapid reversal of anticoagulation by means of prothrombin complex concentrates (PCC, 100 IU/kg) at the end of the 3 h MCAO period, but prior to rt-PA administration, neutralized the exacerbated risk of HT as compared to sham-treated controls (3.8±0.7 µl vs. 15.0±3.8, p<0.001).
Conclusion: In view of the vastly increased risk of HT, it seems to be justified to withhold tPA therapy in effectively anticoagulated patients with acute ischemic stroke. The rapid reversal of anticoagulation with PCC prior to tPA application reduces the risk attributed to warfarin pretreatment and may constitute an interesting therapeutic option.
Changes in vitamin D serum levels have been associated with inflammatory diseases, such as inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis (MS), atherosclerosis, or asthma. Genome- and transcriptome-wide studies indicate that vitamin D signaling modulates many inflammatory responses on several levels. This includes (i) the regulation of the expression of genes which generate pro-inflammatory mediators, such as cyclooxygenases or 5-lipoxygenase, (ii) the interference with transcription factors, such as NF-κB, which regulate the expression of inflammatory genes and (iii) the activation of signaling cascades, such as MAP kinases which mediate inflammatory responses. Vitamin D targets various tissues and cell types, a number of which belong to the immune system, such as monocytes/macrophages, dendritic cells (DCs) as well as B- and T cells, leading to individual responses of each cell type. One hallmark of these specific vitamin D effects is the cell-type specific regulation of genes involved in the regulation of inflammatory processes and the interplay between vitamin D signaling and other signaling cascades involved in inflammation. An important task in the near future will be the elucidation of the regulatory mechanisms that are involved in the regulation of inflammatory responses by vitamin D on the molecular level by the use of techniques such as chromatin immunoprecipitation (ChIP), ChIP-seq, and FAIRE-seq.
Gout is the most common arthritic disease in human but was long neglected and therapeutic options are not satisfying. However, with the recent approval of the urate transporter inhibitor lesinurad, gout treatment has experienced a major innovation. Here we show that lesinurad possesses considerable modulatory potency on peroxisome proliferator-activated receptor γ (PPARγ). Since gout has a strong association with metabolic diseases such as type 2 diabetes, this side-activity appears as very valuable contributing factor to the clinical efficacy profile of lesinurad. Importantly, despite robustly activating PPARγ in vitro, lesinurad lacked adipogenic activity, which seems due to differential coactivator recruitment and is characterized as selective PPARγ modulator (sPPARγM).
Background: The alpha-7 nicotinic acetylcholine receptor (alpha 7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha 7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha 7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha 7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance: The results reported here demonstrate a hitherto unknown relationship between the alpha 7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.
UPF1 regulates myeloid cell functions and S100A9 expression by the hnRNP E2/miRNA-328 balance
(2016)
UPF1 is a key player in nonsense mediated mRNA decay (NMD) but also involved in posttranscriptional gene regulation. In this study we found that UPF1 regulates the expression of genes with functions in inflammation and myeloid cell differentiation via hnRNP E2. The majority of the UPF1-regulated genes identified in monocytic cells contain a binding site for hnRNP E2 within 5′ UTR located introns with hnRNP E2 acting here as splicing regulator. We found that miRNA-328 which is significantly induced during monocytic cell differentiation acts independently from its gene silencing function as RNA decoy for hnRNP E2. One representative gene controlled by the hnRNP E2/miRNA-328 balance is S100A9 which plays an important role in cell differentiation and oxidative stress response of monocytes. Induction of miRNA-328 expression during cell differentiation antagonizes the blockade by hnRNP E2 which results in the upregulation of CD11b expression and ROS production in monocytic cells. Taken together, our data indicate that upregulation of miR-328 is responsible for the induction of hnRNP E2 target genes during myeloid cell differentiation.
The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.
Curcumin, the active constituent of Curcuma longa L. (family Zingiberaceae), has gained increasing interest because of its anti-cancer, anti-inflammatory, anti-diabetic, and anti-rheumatic properties associated with good tolerability and safety up to very high doses of 12 g. Nanoscaled micellar formulations on the base of Tween 80 represent a promising strategy to overcome its low oral bioavailability. We therefore aimed to investigate the uptake and transepithelial transport of native curcumin (CUR) vs. a nanoscaled micellar formulation (Sol-CUR) in a Caco-2 cell model. Sol-CUR afforded a higher flux than CUR (39.23 vs. 4.98 μg min−1 cm−2, respectively). This resulted in a higher Papp value of 2.11 × 10−6 cm/s for Sol-CUR compared to a Papp value of 0.56 × 10−6 cm/s for CUR. Accordingly a nearly 9.5 fold higher amount of curcumin was detected on the basolateral side at the end of the transport experiments after 180 min with Sol-CUR compared to CUR. The determined 3.8-fold improvement in the permeability of curcumin is in agreement with an up to 185-fold increase in the AUC of curcumin observed in humans following the oral administration of the nanoscaled micellar formulation compared to native curcumin. The present study demonstrates that the enhanced oral bioavailability of micellar curcumin formulations is likely a result of enhanced absorption into and increased transport through small intestinal epithelial cells.
Leukotrienes (LTs) are pro-inflammatory lipid mediators that belong to the group of eicosanoids, which are oxygenated metabolites of one common precursor, the aracidonic acid (AA). This polyunsaturated fatty acid is esterified at the sn-2 position of cellular membrane phospholipids and can be released by cytosolic phospholipase A2 alpha (cPLA2alpha) enzymatic deacylation. AA can be converted into LTs by the catalytic reaction of 5-lipoxygenase (5-LO). Enzymatic activation of cPLA2alpha as well as of 5-LO is regulated by similar determinants. In response to cellular stimuli that elevate the intracellular Ca2+ level and/or activate MAP kinase pathways, cPLA2alpha and 5-LO comigrate from a soluble cell compartment (mainly the cytosol) to the nuclear membrane, where AA is released und converted into LTs. LTs play a significant role in promoting inflammatory reactions and immune processes. They have been shown to be released from leukocytes in response to bacterial and viral infections and substantially contribute to an effective immune reaction for host defense. Innate immune pathogen recognition is mediated to a substantial part by the Toll-like receptor (TLR) family. So far, 10 human TLR subtypes have been identified, all of which detect distinct highly conserved microbial structures and trigger the induction of signaling pathways that lead to the expression of numerous immune and inflammatory genes. TLR signaling culminates in the activation NF-kappaB and/or MAP kinases, which as well are known to be involved in the regulation of cellular LT biosynthesis. In this regard, it seemed conceivable that the release of LTs might be regulated by TLR activation. Present studies were undertaken in order to verify and characterize a possible influence of TLR activation on the LT biosynthesis, and furthermore to identify the involved signaling pathways and underlying mechanisms. First experiments revealed that pre-incubation of differentiated Mono Mac 6 (MM6) cells with a TLR4 ligand, a TLR5 ligand, as well as with different TLR2 ligands led to an about 2-fold enhancement of Ca2+ ionophore induced LT biosynthesis. Ligands of other TLR subtypes did not show any influence. These observations could also be confirmed in primary human monocytes stimulated with ionophore or fMLP. With focus on TLR2 ligands, further studies were carried out to characterize the observed enhancement of LT biosynthesis in MM6 cells. It was demonstrated that the extent of LT formation was dependent on the ligand concentration used, but was also dependent on the duration of pre-incubation. Ligand pre-incubation of 15 minutes was optimal to maximally enhance LT formation and further prolongation of pre-incubation decreased LT formation again. Moreover, simultaneous addition of TLR2 ligands with ionophore did also not enhance LT formation. These results indicated that TLR2 ligands seemed to prime human monocytes for an enhanced response upon ionophore stimulation, but did not act as costimuli, which per se were not capable of directly stimulating the biosynthesis of LTs. To analyze the underlying mechanism, the impact of TLR2 ligands on the two key enzymes of the LT biosynthesis pathway, cPLA2alpha and 5-LO, was investigated. In this regard, 5-LO could not been shown to be positively regulated by TLR ligand priming. Neither a direct stimulation, nor an enhancement of 5-LO activity by TLR ligands was detectable in MM6 cells. Similarly, TLR2 ligands did also not enhance ionophore induced 5-LO translocation to the nuclear membrane. However, it was shown that TLR2 ligands enhanced ionophore induced release of AA in MM6 cells, which occurred with a similar time course as LT formation, displaying a maximum at 10 minutes of pre-incubation. A direct stimulation of AA release, however, could not been detected. Inhibitor studies revealed cPLA2alpha to be essential for AA release in TLR2 ligand primed, ionophore stimulated MM6 cells, but also sPLA2 was found to be involved. However, the priming effect of TLR2 ligands was mediated exclusively by cPLA2alpha. Western Blot analyses revealed that p38 MAP kinase, as well as ERK1/2, are activated in MM6 cells in response to TLR2 ligands, and also Ser-505 phosphorylation of cPLA2alpha was detected, which is known to be mediated by MAP kinases and to increase cPLA2alpha activity in vitro. Maximal cPLA2alpha phosphorylation occurred after 5-10 minutes of TLR2 ligand incubation, slightly preceding maximal AA release at 10 minutes and maximal LT formation at 15 minutes of priming. The combined use of a specific p38 MAPK inhibitor with an inhibitor of the ERK1/2 signaling pathway resulted in a complete prevention of cPLA2alpha phosphorylation and TLR2 ligand mediated enhancement of AA release. Thus, both MAPK pathways seem to play a role for TLR2 ligand mediated priming effects on the release of AA. An impact of other kinases such as Mnk-1 and CamKII, which can also regulate cPLA2alpha by phosphorylation, was excluded. Finally, an anti-hTLR2 antibody significantly reduced enhanced AA release, confirming the priming effects to be dependent on TLR2 activation. In summary, it was concluded that the increase of LT biosynthesis by TLR2 ligand priming is considerably due to an enhanced cellular AA supply, which arises from a MAPK mediated phosphorylation and up-regulation of cPLA2alpha. TLR dependent enhancement of LT biosynthesis represents an interesting link between activation of innate immune receptors and the rapid formation of pro-inflammatory lipid mediators. On the one hand, this support the role of LTs in host defence and infectious diseases, but may also be relevant in pathophysiological processes, which involve TLRs as well as LTs, as it has been shown for the pathogenesis of atherosclerosis or allergic diseases.
Suicide genes have been broadly used in gene therapy. They can serve as safety tools for conditional elimination of infused cells or for directed tumor therapy. To date, the Herpes simplex virus thymidine kinase/ ganciclovir (HSVtk/GCV) system is the most prominent and the most widely used suicidegene/prodrug combination. Despite its promising performance, the system displays limitations, which include relatively slow killing kinetics and toxicity of the prodrug GCV. Consequently, several groups have either developed new suicide-gene/prodrug combinations or attempted to improve the established HSVtk/GCV suicide system. The present study also aimed towards optimization of the HSVtk/GCV system. To do so, a novel, codon-optimized point mutant (A168H) of HSVtk was developed. The novel mutant was named TK.007. It was extensively tested for its efficiency in two relevant settings: (1) control of severe graft-versus-host disease (GvHD) after adoptive immunotherapy with Tlymphocytes, and (2) direct elimination of targeted tumor cells. TK.007 was compared to the broadly used wild-type, splice-corrected scHSVtk and to a codon-optimized HSVtk (coHSVtk) not bearing the above point mutation. (1) For experiments related to the adoptive immunotherapy approach, HSVtkvariants were expressed from a γ-retroviral MP71 vector as a fusion construct with the selection and marker gene tCD34. Expression levels for TK.007 in transduced lymphoid and myeloid cell lines were significantly higher at initial transduction and over a 12 week period compared to the commonly used scHSVtk and coHSVtk indicating reduced toxicity of TK.007. Killing kinetics of transduced cell lines (PM1 and K562) and primary human T cells were significantly faster for TK.007 in comparison to scHSVtk and coHSVtk in vitro. In vivo-functionality of TK.007 was assessed in an allogeneic transplantation model. T cells derived from C57BL/6J.Ly5.1 donor mice were transduced with MP71 vectors expressing scHSVtk or TK.007. Transduced cells were selected and transplanted into Balb/c Rag2-/- γ-/- immune-deficient recipient mice. Acute, severe GvHD occurred and was effectively abrogated in all mice transplanted with TK.007- transduced T cells, and in five out of six mice transplanted with scHSVtk-transduced cells. In a slightly modified quantitative allogeneic transplantation mouse model, significantly faster and more efficient in vivo killing was demonstrated for TK.007 as compared to scHSVtk, especially at low doses of GCV. (2) In order to assess TK.007 functionality in cells derived from solid tumors, HSVtk-variants were expressed from lentiviral gene ontology (LeGO) vectors in combination with an eGFP/neo-opt selection cassette. Transduced and selected tumor cell lines that derived from several tissues were eliminated at significantly lower GCV doses and to higher extents when transduced with TK.007 compared to scHSVtk. Moreover, a significantly stronger bystander effect of TK.007 was demonstrated. The superior in vitro efficiency of TK.007 was confirmed in an in vivo subcutaneous xenograft mouse model for glioblastoma in NOD/SCID mice. Mice transplanted with TK.007 transduced cells stayed tumor-free after treatment with different GCV-doses. On the contrary, mice of the scHSVtk group either demonstrated only transiently reduced tumor growth in the low-dose GCV group (10 mg/kg) compared to the control groups or suffered from relatively fast relapses after initial tumor shrinking in the standarddose (50 mg/kg) GCV group. As a result, all mice in the scHSVtk group died from vigorous tumor growth. In summary, in two different applications for suicide gene therapy the present study has demonstrated superior functional performance of the novel suicide gene TK.007 as compared to the broadly used wild-type scHSVtk. Differences became particularly pronounced at low doses of GCV. It can be concluded that the new TK.007-gene represents a promising alternative to the commonly used scHSVtk for gene therapeutic applications.
Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.
The vacuolar-type H+-ATPase (v-ATPase) is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells (ECs), in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.
Acute myeloid leukemia (AML) is a clonal malignancy of hematopoietic stem cells (HSCs) characterized by expansion of myeloid blasts in the bone marrow. It has been shown that autophagy is a degradative process, which delivers cytoplasmic components to lysosomes to prevent malignant transformation by maintaining HSC integrity. Besides its function as a bulk degradation machinery to recycle cytoplasmic components during limited energy supply, autophagy also serves as an intracellular quality control mechanism. Selective autophagy requires autophagy receptors such as p62 to specifically bridge the targeted cargos into autophagosomes. p62 is known as a central signaling hub involved in pro-oncogenic signaling pathways and autophagic degradation pathways. However, little is known about the role of p62 as a selective autophagy receptor in AML. This study aims to elucidate the precise function of p62 as an autophagy receptor in leukemia development and maintenance.
In silico analysis revealed that high p62 expression was significantly associated with poor overall survival of adult patients with de novo AML, suggesting that p62 may promote leukemia maintenance. To address the functional role of p62 in leukemia, genome editing by CRISPR/Cas9 was used to knockout p62 in four human AML cell lines. Importantly, p62 loss reduced cell proliferation in all four cell lines. This observation could be transferred to a murine leukemia cell model in which leukemic transformation of lineage-depleted bone marrow (ldMBM) cells was induced by overexpression of the human transcriptional coactivator MN1. Knockdown of p62 by shRNA in MN1-driven leukemia cells impaired proliferation and decreased colony forming ability without altering apoptosis. This indicates that p62 is crucial for leukemia proliferation in vitro. To further characterize the role of p62 in leukemia development and maintenance a murine AML transplantation model was established. Therefore, ldMBM cells isolated from WT and p62-/- mice were transduced with MN1 and transplanted into lethally irradiated mice. As expected, all mice developed fatal myeloid proliferation. Notably, p62 loss in MN1-driven leukemia significantly prolonged survival in mice and caused a more immature phenotype. Consistent with the in vitro results, ex vivo analysis of p62-/- leukemic cells displayed decreased colony-forming ability, although p62 loss did not affect composition and function of HSCs. Moreover, re-transplantation of primary MN1-driven leukemia cells attenuated leukemia progression upon p62 loss. These findings support a decisive role of p62 in leukemia development and maintenance.
To gain molecular insight into the function of p62 during myeloid transformation an interactome analysis of murine MN1-driven leukemia cells was performed. This revealed first that p62 predominantly interacts with mitochondrial proteins and second that inhibition of autophagic degradation causes accumulation of p62-bound mitochondria. This leads to the first assumption that loss of p62 may provoke mitochondrial accumulation with increasing mitochondrial damage and second that p62 may mediate degradation of mitochondria by mitophagy. Indeed, in the absence of p62, accumulation of dysfunctional mitochondria was detected by morphological changes of the mitochondria, increased mitochondrial ROS and impaired mitochondrial respiration capacity. Furthermore, induction of PINK1/Parkin-independent mitophagy revealed that loss of p62 caused impaired degradation of mitochondrial proteins and reduced translocation of damaged mitochondria into autophagosomes. Taken together, p62 is required for effective degradation of dysfunctional mitochondria by mitophagy in AML.
Due to the fact that p62 is a multifunctional protein, rescue experiments with different mutants of p62 were performed to clarify if p62-mediated mitophagy contributes to leukemia proliferation. Notably, the autophagy-deficient mutant (disabled to bind autophagosomes) reduced cell growth and colony-forming ability to the same extent as knockdown of p62, as the clustering-deficient mutant (disabled to form aggregates) displayed an intermediate phenotype. Strikingly, only the autophagy-deficient mutant failed to rescue mitophagy.
In conclusion, this study demonstrates the prominent role of p62 as a selective autophagy receptor for mitochondrial quality control which contributes to leukemia development and maintenance. Therefore, targeting selective autophagy opens new venues in the treatment of AML.
The role of the Ca2+-dependent protease calpain in the diabetes-associated platelet hyperreactivity
(2012)
Platelets from diabetic patients are characterised by hyperreactivity resulting in exaggerated adhesion, aggregation and thrombus formation which contribute to the development of cardiovascular complications known to be one of the main causes of diabetes-related mortality. One of the mechanisms suggested to be involved in the diabetes-related platelet hyperactivation is the increased [Ca2+]i which leads to the overactivation of Ca2+-dependent proteases, the calpains. Among the calpain isoforms expressed in platelets the two ubquitiously expressed μ- and m-calpain are thought to play an important role in physiological and pathophysiological processes. Particularly μ-calpain is known to be involved in many steps of physiological platelet activation such as aggregation, adhesion, secretion, and signalling. However, we could show that diabetes was associated with an enhanced activation of both μ- and m-calpain in platelets
In the first part of the study we focussed on the characterization of the molecular mechanism regulating calpain activity. Indeed, although Ca2+ is considered to be the main regulator of the proteolytic activity of the conventional calpains, other mechanisms such as the presence of phospholipids and phosphorylation have been reported to affect their activity. Since most studies reported the phosphorylation of m-calpain we were interested to see whether μ-calpain activity might be also affected by phosphorylation. We could show that the activity of μ-calpain was enhanced by the PKC activator PMA suggesting its possible regulation by phosphorylation. However, whether PKC directly targeted μ-calpain remains unclear. Given that substrate recognition is important for a protease to process its substrate and since no common consensus could be attributed to calpain substrates, our next interest was to understand the mechanism regulating the recognition of its substrates by calpain. Since phosphorylation has been reported to protect different proteins from calpain degradation we investigated whether the calpain substrate CD31 could be phosphorylated in platelets and whether this could affect its recognition by calpain. Although we could show that the tyrosine phosphorylation of CD31 was increased after activation of platelets by thrombin and that this effect was attenuated in platelets from diabetic patients, tyrosine phosphorylation of CD31 seemed to have no effect on its sensitivity to calpain-mediated proteolysis.
After the analysis of the mechanism regulating calpain activity as well as its interaction with its substrates, our next interest was the identification of new calpain substrates in platelets. Since a previous study from our group showed that PPARγ agonists could indirectly reverse the diabetes-associated calpain activation we performed DIGE analysis of platelet samples from diabetic patients before and after PPARγ agonist treatment. Using this approach we could identify four novel calpain substrates in platelets: Integrin-linked kinase (ILK), α parvin, CLP36 and septin-5. Next, we assessed the effect of calpain-mediated cleavage on the function of these newly identified proteins. We could show that μ-calpain was essential for the dissociation of ILK from the IPP complex and its activation while m-calpain-mediated cleavage led to its cleavage and inactivation. Functionally, we also showed that μ-calpain was involved in platelet adhesion while m-calpain was important for spreading.
The next protein we analysed was septin-5, a small GTPase known to regulate platelet degranulation by association with other septins and syntaxin-4. We found that the interaction between septin-5 and syntaxin-4 was inhibitory for platelet degranulation. We could demonstrate that the μ-calpain-mediated cleavage dissociated septin-5 from syntaxin 4 and led to increased secretion of platelet α-granules. Next, we investigated the in vivo role of calpain in the diabetes-associated platelet hyperreactivity. We induced diabetes in mice and could reproduce calpain activation in platelets such as that found in human. Indeed, calpain activation in murine platelets also led to the cleavage of several calpain substrates including ILK and septin-5. Moreover, platelets from diabetic mice demonstrated an increased aggregation and thrombus formation in vivo. Treatment of the animals with the calpain inhibitor A-705253 (30 mg/kg/day for 10 days) significantly restored platelet function and substrate cleavage. In conclusion, in this part of the study, we could show that the increased calpain-dependent α-granule secretion and platelet adhesion may account for the enhanced vascular proliferation and thrombus formation in diabetes and calpain inhibition represents a promising way to prevent atherothrombosis development.
In the last part of the study we analysed another enzyme known to play a crucial role in diabetes, the AMPK which is an energy-sensing kinase known to be impaired in diabetes. We could show that the two catalytic subunits AMPK α1 and α2 are expressed in platelets. The AMPKα2 seemed to be the subunit involved in platelet activation since AMPKα2-deficient mice demonstrated a defect in clot retraction and the stabilization of the thrombus while the animals showed a normal bleeding time. Mechanistically, we showed in platelets that the upstream kinase of AMPKα2 is LKB1 which was activated by thrombin stimulation via a PI-3K-dependent pathway. AMPKα2 then phosphorylated the Src-family kinase Fyn, which is responsible for the phosphorylation of its substrate β3 integrin on Tyr747. These data indicate that AMPKα2, by affecting Fyn phosphorylation and activity, plays a key role in platelet αIIbβ3 integrin signalling, leading to clot retraction and thrombus stability. Although the effect of diabetes in the AMPK-dependent pathway could not be investigated we assume that the dysregulation of this pathway may account for the thrombus destabilization and enhanced embolization encountered in diabetes.
Alzheimer’s disease is a chronic neurodegenerative disease that causes problems with memory, thinking and behavior. The pathophysiological hallmarks of AD are extracellular senile plaques and intracellular neurofibrillary tangles. Amyloid plaques mainly contain the amyloid-β (Aβ) peptide, which appears as a cleavage product of the APP. APP is a type I transmembrane protein with a large extracellular domain and a short cytoplasmic tail. It is expressed in variety of tissues e.g. in neuronal tissue (brain, spinal cord, retina), and non-neuronal tissues (kidney, lung, pancreas, prostate gland, and thyroid gland) (Dawkins and Small, 2014). APP has been studied because of its link to AD, however, its role in normal brain function is poorly understood. APP is processed by two different pathways, amyloidogenic pathway and non-amyloidogenic pathway. In physiological condition, the majority of APP is processed via the non-amyloidogenic, thus leading to the generation of the secreted N-terminal APP processing product sAPPα. sAPPα is formed due to the cleavage of APP by α-secretase. In previous studies, our group has shown that sAPPα produce potent neuroprotective effect by altering gene expression, as well as by antagonizing several different types of neurotoxic stress stimuli (Copanaki et al., 2010; Kögel et al., 2003, 2005; Milosch et al., 2014). Several studies have shown that protein degradation is reduced in AD (Hong et al., 2014; Lipinski et al., 2010) but the role of APP and its cleavage products in protein degradation is still unknown. This thesis discusses about the physiological functions of APP in neuroprotection and protein homeostasis.
In the first part of the thesis (Section 4.1 - 4.4), the neuroprotective properties of yeast derived sAPPα and E1 (N-terminal domain of sAPPα) were investigated under serum and glucose deprivation conditions. In previous work, it was shown that recombinant sAPPα evoked a significant decrease in serum deprivation triggered cell death in human SH-SY5Y neuroblastoma cells and mouse embryonic fibroblast MEF cells. It was also observed that sAPPα induces the phosphorylation of Akt which leads to neuroprotection (Milosch et al., 2014). This study investigated whether this neuroprotection is associated with altered expression of downstream intracellular Akt targets such as FoxO, Bim, Bcl-xL and Mcl-1 under stress conditions. Here it was shown that sAPPα prevents activation and nuclear translocation of FoxO. FoxO act as a transcription factor for different proapoptotic genes such as Bim. It was also observed that Bim protein and mRNA expression was significantly reduced with sAPPα and E1 treatment. The expression of antiapoptotic protiens such as Bcl-xL and Mcl-1 were also examined and it was observed that sAPPα and E1 increases expression of both these proteins. Furthermore, it was previously demonstrated that uncleaved holo-APP functionally cooperates with sAPPα to activate Akt and provide neuroprotection (Milosch et al., 2014). Therefore, to investigate the function of the APP in sAPPα regulated Akt downstream proteins expressions, MEF APP KO cells were used. E1 and sAPPα only showed neuroprotective modulatory effect on these Akt downstream targets in MEF wt cells, but not in APP KO cells. In addition, sAPPα also showed neuroprotection in primary wt hippocampal neurons under trophic factor deprivation. Cellular fractionation experiments were also done to determine the role of sAPPα in cytochrome c release from mitochondria. It was observed that sAPPα treatment can inhibit mitochondrial cytochrome c release in wt MEF cells.
The second part of the thesis (Section 4.5 - 4.9) discusses about the role of sAPPα in protein homeostasis. It was observed that sAPPα prevents proteotoxic stress induced BAG3 protein expression in SH-SY5Y and MEF cells. This was also observed in mRNA levels which indicate a transcriptional regulation. Furthermore, treatment with sAPPα was also shown to decrease aggresomes formation. Aggresomes are perinuclear aggregates which are formed due to accumulation of damaged and misfolded proteins and BAG3 plays important role in their formation and the transport of degradation prone proteins into these structures. The analysis of proteasomal activity showed a reduced accumulation of proteasomal substrate d2 by sAPPα under proteasomal stress. In proteasomal activity assay, sAPPα was shown to increase the degradation of proteasomal substrate SUC-LLVY-AMC and the fluorigenic signal was measured spectrophotometrically. The sAPPβ fragment which is generated via the amyloidogenic pathway was also examined for its role in BAG3 expression and proteasomal degradation. sAPPβ, which has almost similar structure as sAPPα, only 17 amino acids at the C-terminus is missing, was failed to modulate BAG3 expression and proteostasis. This indicates that these biological effects are highly specific for sAPPα.
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Immune cells are key players in several physiological and pathophysiological events such as acute and chronic inflammation, atherosclerosis and cancer. Especially in acute inflammation, macrophages are indispensable for the switch from the acute inflammatory phase to the resolution phase. Not only the phagocytosis of apoptotic cells, but especially the surrounding cytokines and mediators are able to switch macrophage polarization from inflammatory- to anti-inflammatory phenotypes. Within this cytokine environment, sphingosine-1-phosphate (S1P) plays an important role for immune cell activation, polarization and migration.
The liver as the biggest endocrine gland of the human body plays a central role in many metabolic pathways such as detoxification, storage of carbohydrates and distribution of lipids. As the liver receives blood supply from the gut by the portal vein, liver cells are often challenged with high concentrations of nutrients and components of our commensal microbiota. Therefore, the immune system of the liver induces a tolerant state, meaning no or low inflammatory reactions to those constant stimuli. Yet, as various pathogens target the liver, the hepatic immune system also needs the capability to induce strong immune responses quickly. Chronical damage to the liver, which can be caused by alcohol, pathogens or toxins, might lead to liver cirrhosis, where the amount of functional liver tissue is decreased dramatically. This pathology can worsen and lead to acute-on-chronic liver failure, whose high mortality is due to high inflammation and multi-organ failure. Interleukin-7 is a cytokine known for its pro-survival functions especially in lymphopoiesis. However, it is also very important for maintenance of mature immune cells in the liver. As mouse experiments have demonstrated an induction of Interleukin-7 in the liver as a response to bacterial lipopolysaccharide, we aimed to characterize the role of Interleukin-7 in hepatic immunoregulation in both health and disease.
The experiments were mostly based on in vitro approaches. Induction of Interleukin-7 in liver cells was analyzed using ELISA, quantitative PCR, and Immunoblotting. Knockdown of signal transduction components was performed by siRNA transfection. Primary immune cells isolated from healthy donor buffy coat were studied for their ability to respond to Interleukin-7. Activation of downstream signal transduction was assessed by Immunoblotting. Functional consequences of Interleukin-7 signaling, such as alterations in cellular metabolism, cellular survival and endotoxin tolerance, were studied in monocyte-derived macrophages. Finally, serum concentrations of Interleukin-7 and frequencies of Interleukin-7 receptor positive immune cells were quantified in patients with compensated or decompensated liver cirrhosis or acute-on-chronic liver failure.
Interleukin-7 expression could be observed in human hepatic cell lines and primary hepatic sinusoidal endothelial cells when stimulated with IFNα or IFNγ, but not IFNλ. IRF-1 was identified as a key regulator of Interleukin-7 expression, as its transcription, translation and nuclear translocation were induced and enhanced upon IFNα or IFNγ, but not IFNλ treatment. We identified LPS-primed macrophages as innate immune target cells of Interleukin-7, which responded by an inhibitory phosphorylation of GSK3. This signal transduction led to enhanced production of pro-inflammatory cytokines and abolished endotoxin tolerance. In parallel, cellular fitness was reduced as demonstrated by reduced intracellular ATP concentration and intracellular WST-1 staining. Finally, we could identify components of the in vitro signal transduction also in liver cirrhosis patients. However, Interleukin-7 serum concentrations were significantly in liver cirrhosis patients compared to healthy controls. In addition, the frequencies of Interleukin-7 receptor positive immune cell populations differed in patients and controls.
We identify Interleukin-7 as a pro-inflammatory cytokine in hepatic immunoregulation. It is part of a cascade where its induction is regulated by type I and type II Interferons and mainly restricted by the presence of IRF1. We demonstrate the importance of Interleukin-7 also for innate immune cells, where the abolishment of endotoxin tolerance may provide an interesting strategy of liver cirrhosis patients. In addition, reduced viability of macrophages in response to Interleukin-7 is a striking contrast to the well-described survival functions in lymphocytes. The decrease of serum Interleukin-7 levels and alterations of Interleukin-7 receptor positive immune cell populations suggest an important role for Interleukin-7 also in the diseased liver. Due to the identified mechanisms of action, Interleukin-7 may be an interesting candidate for immunotherapeutic approaches of liver cirrhosis and acute-on-chronic liver failure.
First paragraph (this article has no abstract) Persistent stimulation of nociceptors results in sensitization of nociceptive sensory neurons, which is associated with hyperalgesia and allodynia. The release of NO and subsequent synthesis of cGMP in the spinal cord are involved in this process. cGMP-dependent protein kinase I (PKG-I) has been suggested to act as a downstream target of cGMP, but its exact role in nociception hadn't been characterized yet. To further evaluate the NO/cGMP/PKG-I pathway in nociception we assessed the effects of PKG-I inhibiton and activaton in the rat formalin assay and analyzed the nociceptive behavior of PKG-I-/- mice. Open access article.
Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
Microtubule-targeting agents (MTAs) are the most widely used chemotherapeutic drugs. Pretubulysin (PT), a biosynthetic precursor of the myxobacterial tubulysins, was recently identified as a novel MTA. Besides its strong anti-tumoral activities, PT attenuates tumor angiogenesis, exerts anti-vascular actions on tumor vessels and decreases cancer metastasis formation in vivo. The aim of the present study was to analyze the impact of PT on the interaction of endothelial and tumor cells in vitro to gain insights into the mechanism underlying its anti-metastatic effect. The influence of PT on tumor cell adhesion and transmigration onto/through the endothelium as well as its influence on cell adhesion molecules and the chemokine system CXCL12/CXCR4 was investigated. Treatment of human endothelial cells with PT increased the adhesion of breast cancer cells to the endothelial monolayer, whereas their transmigration through the endothelium was strongly reduced. Interestingly, the PT-induced upregulation of ICAM-1, VCAM-1 and CXCL12 were dispensable for the PT-evoked tumor cell adhesion. Tumor cells preferred to adhere to collagen exposed within PT-triggered endothelial gaps via β1-integrins on the tumor cell surface. Taken together, our study provides, at least in part, an explanation for the anti-metastatic potential of PT.
GProteingekoppelte Rezeptoren (GPCRs) stellen eine der größten in der Natur vorkommenden Proteinfamilien dar (Watson and Arkinstall, 1994). GPCRs sind plasmamembranständige Proteine, die mit heterotrimären GProteinen interagieren und eine Vielzahl an Signaltransduktionswegen aktivieren. Trotz der strukturellen Vielfalt der an GPCRs angreifenden Liganden stimulieren die meisten GPCRs nur eine begrenzte Anzahl strukturell sehr ähnlicher GProteine (Hedin et al., 1993; Conklin and Bourne, 1993). Die Aufklärung der molekularen Mechanismen, die dieser Rezeptor/GProteinKopplungsselektivität zugrunde liegen, ist von fundamentaler Wichtigkeit für das Verständnis zellulärer Signaltransduktion. Ausführliche StrukturFunktionsanalysen verschiedener Neurotransmitter rezeptoren, einschließlich der Muskarinrezeptoren (Wess, 1996) und adrenergen Rezeptoren (Dohlman et al., 1991; Savarese and Fraser, 1992; Strader et al., 1994), haben einen beträchtlichen Beitrag zur Identifizierung der strukturellen Elemente, die für die GProteinKopplungsselektivität dieser Rezeptorgruppe verantwortlich sind, geleistet. Im Gegensatz dazu ist bisher noch weitgehend ungeklärt, welche molekularen Mechanismen der Kopplungsselektivität von GPCRs, die durch Peptidliganden aktiviert werden, zugrunde liegen. Das Ziel dieser Arbeit war daher, molekulare Grundlagen der GProtein Kopplungsselektivität von PeptidGPCRs näher zu untersuchen und aufzuklären. Die Vasopressinrezeptorfamilie unterscheidet sich von nahezu allen anderen PeptidGPCRs darin, daß die einzelnen Rezeptorsubtypen deutlich unterschiedliche GProtein Kopplungspräferenzen aufweisen. Die V1a und V1bVasopressinrezeptoren stimulieren selektiv GProteine der Gq/11 Familie, was zur Aktivierung von PhospholipaseCbeta-Isomeren führt. Im Gegensatz dazu koppelt der V2Vasopressinrezeptor vornehmlich an das GProtein G s , was in einem Anstieg an intrazellulärem cAMP resultiert. Daher stellen die Vasopressinrezeptorsubtypen ein attraktives Modellsystem zum Studium der Peptid GPCRRezeptordomänen, die für die selektive GProteinAktivierung verantwortlich sind, dar. Als Modellsystem für diese Arbeit diente primär der V2Vasopressinrezeptor. Molekulare Faktoren, die die Gs Kopplungsselektivität des V2 Vasopressinrezeptors bestimmen. Eine frühere Studie zeigte, daß die Gegenwart der V1aRezeptorsequenz in der zweiten intrazellulären (i2) Schleife notwendig war, um den Wildtyp V1a und V1a/V2 Rezeptorchimären effizient an Gq/11 Proteine zu koppeln (Liu and Wess, 1996). Effiziente Interaktionen zwischen Wildtyp V2 oder V1a/V2Rezeptorchimären und dem GProtein G s waren hingegen hauptsächlich von V2Rezeptorsequenzen in der dritten intrazellulären (i3) Schleife abhängig. Um die molekularen Grundlagen der Gs Kopplungsselektivität des V2Rezeptors näher zu untersuchen, wurden zunächst klassische Mutagenesetechniken (zielgerichtete Mutagenese'') angewandt. Definierte V2Rezeptorsegmente (oder einzelne Aminosäuren) wurden in den V1aRezeptor transferiert, und die resultierenden HybridVasopressinrezeptoren wurden anschließend in funktionellen Studien auf ihre Fähigkeit, hormonabhängig intrazelluläre cAMP Konzentrationen zu steigern (G s vermittelt), getestet. Diese Strategie schien besonders geeignet, da die Aktivierung des V1aWildtyprezeptors nahezu keine Auswirkungen auf intrazelluläre cAMPSpiegel hat. Wie bereits erwähnt, ist die effiziente Kopplung des V2Rezeptors an das Gs Protein vornehmlich von V2Rezeptorsequenzen in der i3Schleife abhängig (Liu and Wess, 1996). Eine V1aRezeptormutante, deren i3Schleife durch die homologe V2 Rezeptorsequenz ersetzt worden war, war in der Lage, effizient mit Gs zu interagieren. Die Fähigkeit dieser Rezeptormutante, Gs zu aktivieren, war jedoch im Vergleich zum V2Wildtyprezeptor vermindert. Diese Beobachtung ließ die Vermutung zu, daß noch andere intrazelluläre V2Rezeptordomänen zur optimalen Gs Kopplung notwendig sind. Daher wurde zunächst eine Reihe von V1a/V2Rezeptorchimären erzeugt, die den Beitrag der zweiten (i2) und vierten intrazellulären (i4) Rezeptordomäne zur V2 Rezeptor/G s Kopplungsselektivität klären sollten. Funktionelle Untersuchungen der resultierenden HybridRezeptormutanten in Säugetierzellen (COS7) zeigten, daß ein kurzes Segment im Nterminalen Abschnitt der i4Domäne einen deutlichen Beitrag zur V2Rezeptor/G s Kopplungsselektivität leistet. Eine V1aRezeptormutante, welche in der i3Schleife und dem Nterminalen Segment der i4Domäne (Ni4) homologe V2 Rezeptorsequenzen enthielt, zeigte ein funktionelles Profil (EC 50 und E max ), welches mit dem V2Wildtyprezeptor nahezu deckungsgleich war. Anschließend wurden strukturelle Elemente innerhalb der i3Schleife näher untersucht. Funktionelle Analysen zeigten, daß der Nterminale Abschnitt der i3Schleife weitgehend das GProteinKopplungsprofil des V2Rezeptors bestimmt. Eine Reihe von V1aRezeptormutanten wurde erzeugt, in denen kurze Segmente des Nterminalen Bereichs der i3Schleife mit der entsprechenden V2Rezeptorsequenz ausgetauscht wurden. Funktionelle Untersuchungen ergaben, daß ein Aminosäurepaar (Gln225, Val226) und triplet (Phe229, Arg 230, Glu231) am Beginn der i3Schleife des V2 Rezeptors für die effiziente Aktivierung von Gs von entscheidender Bedeutung sind. Durch Punktmutationen in diesem Bereich wurden zwei polare Aminosäuren, Gln225 und Glu231, identifiziert, die für die effiziente V2Rezeptor/G s Interaktion essentiell sind. Untersuchungen mit anderen GPCRKlassen (Dohlman et al., 1991; Savarese and Fraser, 1992; Strader et al., 1994; Wess, 1996) haben ebenfalls gezeigt, daß dem N Terminus der i3Schleife eine besondere Rolle im Rezeptor/GProteinKopplungsprozeß zukommt. In diesen Studien wird berichtet, daß vornehmlich hydrophobe und ungeladene Aminosäuren Schlüsselrollen in der rezeptorvermittelten GProteinAktivierung einnehmen. Die hier beschriebenen Untersuchungen hingegen ergaben, daß zwei polare/geladene Aminosäuren, Gln225 und Glu231, für die V2Rezeptor/G s Kopplung von besonderer Wichtigkeit sind und zeigen daher, daß die Rezeptor/GProtein Kopplungsselektivität nicht auf ausschließlich hydrophoben Wechselwirkungen beruht. Desweiteren konnte beobachtet werden, daß die Länge der i3Schleife die Effizienz, mit der der V2Rezeptor GProteine der Gs Klasse zu aktivieren vermag, beeinflußen kann. Die V1a und V2Rezeptoren weisen unterschiedlich lange i3 Schleifen auf (die i3Schleife des V2Rezeptors ist 13 Aminosäuren kürzer als die des V1aRezeptors). Eine V1aRezeptormutante, deren Nterminaler Abschnitt der i3 Schleife durch homologe V2Rezeptorsequenz ersetzt wurde, konnte deutlich effizienter mit Gs interagieren, wenn der mittlere Abschnitt der i3Schleife um elf Aminosäuren verkürzt wurde. Gleichermaßen konnte die effiziente Kopplung bestimmter V1a/V2Hybridrezeptoren an Gs durch Einfügen von elf Aminosäuren in den zentralen Bereich der i3Schleife deutlich gehemmt werden. Diese Ergebnisse legen nahe, daß der zentrale Bereich der i3Schleife die Rezeptor/GProteinKopplungsselektivität beeinflussen kann, obgleich diese Rezeptordomäne vermutlich nicht direkt mit dem GProtein interagiert. Es ist denkbar, daß die Länge der i3Schleife den Zugang des GProteins zu funktionell wichtigen Rezeptordomänen, z.B. Aminosäuren im Bereich der fünften Transmembrandomäne (TM V) und der i3Schleife, reguliert. Identifizierung einzelner Aminosäuresubstitutionen und Aminosäuredeletionen, die die GProteinKopplungsselektivität des V2Rezeptors beeinflussen: Einsatz von Hefeexpressionstechnologie und zufallsgerichteter Mutagenese (random mutagenesis'') Im zweiten Teil dieser Arbeit wurden Hefe(Saccharomyces cerevisiae) Expressionstechnologien angewandt, um StrukturFunktionsanalysen des V2Rezeptors zu erleichtern und Beschränkungen klassischer Mutagenesetechniken zu überwinden. Der V2Wildtyprezeptor und verschiedene GProteinchimären aus Hefe und SäugetierGalpha Untereinheiten wurden in genetisch modifizierten Hefelinien, deren Zellwachstum von effizienter Rezeptor/GProteinKopplung abhängig war, coexprimiert. In diesem System aktiviert produktive Rezeptor/GProteinKopplung den HefeMAPKinase/Pheromon Signaltransduktionsweg. Dies führt zur Transkription des FUS1HIS3Reportergens und somit zur Expression von His3Protein, was den Histidinauxotrophen (his3) Hefelinien ermöglicht, in histidinfreiem Medium zu wachsen (Pausch et al., 1998). Es konnte gezeigt werden, daß heterolog exprimierte V2Rezeptoren weder mit der HefeGProtein alphaUntereinheit (Gpa1p) noch mit einem mutierten Gpa1Protein, in dem die Cterminalen fünf Aminosäuren gegen homologe Galpha q Sequenz ausgetauscht worden waren (Gq5), effizient interagierten. Im Gegensatz dazu erwies sich die Interaktion zwischen dem V2 Rezeptor und einem mutierten Gpa1Protein, dessen Cterminale fünf Aminosäuren die homologe Galpha s Sequenz enthielten (Gs5), als hocheffizient. Diese Beobachtungen zeigten, daß der V2Rezeptor im Hefesystem sein physiologisches Kopplungsprofil beibehielt. Zur weiteren Validierung des Hefeexpressionssystems wurden die G q/11 gekoppelten M 1 , M 3 und M 5 Muskarinrezeptoren und verschiedene mutierte Vasopressin und M 3 Muskarinrezeptoren mit veränderten funktionellen Eigenschaften heterolog in Hefe exprimiert. Funktionelle Analysen zeigten, daß die Wildtyprezeptoren und die verschiedenen Rezeptormutanten in Hefe und Säugetierzellen ähnliche Phänotypen aufwiesen. Um zu untersuchen, weshalb der V2Rezeptor nicht effizient an GProteine der Gq/11 Familie koppelt, sollte der in Hefe exprimierte V2Rezeptor zufallsgerichteter Mutagenese (random mutagenesis'') unterzogen und Mutanten mit veränderten G ProteinKopplungeigenschaften isoliert werden. Im speziellen wurde die i2Schleife untersucht, da eine frühere Studie gezeigt hatte, daß vornehmlich die i2Schleife des V1a Rezeptors für die V1aRezeptor/G q/11 Kopplungsselektivität verantwortlich ist (Liu and Wess, 1996). Mittels zufallsgerichteter Mutagenesetechnik wurde in Hefe eine Bibliothek von V2Rezeptormutanten erzeugt, deren i2Schleife Mutationen mit einer Mutageneserate von ungefähr 10% (auf der Nukleotidebene) enthielt. Anschließend wurden in einem Selektionsverfahren (screen'') 30 000 V2Rezeptormutanten auf ihre Fähigkeit, mit Gq5 zu interagieren, überprüft. Es konnten vier V2Rezeptormutanten isoliert werden, welche effizient an Gq5 (jedoch nicht an HefeGpa1p) koppelten. Funktionelle Untersuchungen mit diesen und anderen mittels zielgerichteter Mutagenese erzeugter V2Rezeptormutanten zeigten, daß die Substitution einer einzigen Aminosäure (Met145) im zentralen Bereich der i2Schleife beträchtliche Auswirkungen auf die Rezeptor/GProteinKopplungsselektivität hatte. Die Fähigkeit des V2Rezeptors, produktiv mit Gq5 zu interagieren, war von der Anwesenheit relativ großer, hydrophober Aminosäuren wie Leucin und Tryptophan abhängig. Austausch von Met145 mit kleinen Aminosäuren wie Glycin oder Alanin erlaubte dem V2Rezeptor nicht, Gq5 zu aktivieren. Interessanterweise interagierten alle V2Rezeptormutanten, die eine Met145 Punktmutation aufwiesen, mit Gs5 ähnlich effizient wie der V2Wildtyprezeptor. Die Unfähigkeit der V2(Met145Gly) und V2(Met145Ala)Rezeptoren, Gq5 zu aktivieren, beruht daher nicht auf einem Faltungsdefizit. Gleichermaßen basierte die Fähigkeit der V2(Met145Trp) und V2(Met145Leu)Rezeptoren, produktiv an Gq5 zu koppeln, nicht auf der Überexpression von Rezeptorprotein. Diese Ergebnisse zeigen, daß die chemische Eigenschaft der Aminosäure an Position 145 die V2Rezeptor/GProtein Kopplungsselektivität reguliert. Interessanterweise befindet sich in allen anderen Subtypen der Vasopressin/OxytocinRezeptorfamilie (V1a, V1b, und Oxytocin Rezeptoren), welche selektiv an GProteine der G q/11 Klasse gekoppelt sind, ein Leucin an der Stelle, die zu Met145 (V2Rezeptorsequenz) homolog ist. Eine der vier ursprünglich isolierten V2Rezeptormutanten enthielt neben verschiedenen Punktmutationen eine Deletion in Position Met145. In detaillierteren zielgerichteten MutageneseStudien wurden zwei V2Rezeptormutanten erzeugt, die alle drei GProteine (Gq5, Gs5 und Gpa1p) aktivieren konnten. Um zu untersuchen, ob ein generelles Verkürzen der i2Schleife um eine Aminosäure der Grund für die beobachtete Rezeptor/GProteinPromiskuität ist, wurden verschiedene V2Rezeptormutanten erzeugt, in denen einzelne Aminosäuren unmittelbar N und Cterminal von Met145 deletiert worden waren. Funktionelle Untersuchungen ergaben, daß die Deletion einzelner Aminosäuren Nterminal von Met145 (Ile141delta, Cys142delta, Arg143delta oder Pro144delta) in V2Rezeptormutanten resultierte, die nicht mit GProteinen interagieren konnten. RadioligandBindungsstudien zeigten, daß diese V2Rezeptormutanten keine V2Liganden binden konnten, was darauf schließen läßt, daß Deletionen einzelner Aminosäuren Nterminal von Met145 zu mißgefalteten Rezeptoren führen. Die Aminosäuren Ile141Pro144 befinden sich am Beginn der i2Schleife, unmittelbar neben der alphahelikalen zytoplasmatischen Verlängerung der dritten Transmembrandomäne (TM III) in der Nähe des hochkonservierten DRY(H)Motivs. Es ist denkbar, daß Aminosäuren innerhalb des Ile141Pro144Segments mit den zytoplasmatischen Abschnitten von TM III und/oder TM V interagieren und diese Wechselwirkungen die Rezeptorstruktur stabilisieren. Im Gegensatz dazu hatten Deletionen unmittelbar C terminal von Met145 (Leu146delta, Ala147delta, Tyr148delta oder Arg149delta) keinerlei Auswirkungen auf die Funktion des V2Rezeptors. Diese Aminosäuren befinden sich im zentralen Bereich der i2Schleife, der nicht mit den transmembranären Domänen des Rezeptorproteins interagieren kann.
One hallmark of MLL-r leukemia is the highly specific gene expression signature indicative for commonly deregulated target genes. An usual read-out for this transcriptional deregulation is the HOXA gene cluster, where upregulated HOXA genes are detected in MLL-r AML and ALL patients. In case of t(4;11) leukemia, this simple picture becomes challenged, because these patients separate into HOXAhi- and HOXAlo-patients. HOXAlo-patients showed a reduced HOXA gene transcription, but instead overexpressed the homeobox gene IRX1. This transcriptional pattern was associated with a higher relapse rate and worse outcome. Here, we demonstrate that IRX1 binds to the MLL-AF4 complex at target gene promotors and counteract its promotor activating function. In addition, IRX1 induces transcription of HOXB4 and EGR family members. HOXB4 is usually a downstream target of c-KIT, WNT and TPO signaling pathways and necessary for maintaining and expanding in hematopoietic stem cells. EGR proteins control a p21-dependent quiescence program for hematopoietic stem cells. Both IRX1-dependend actions may help t(4;11) leukemia cells to establish a stem cell compartment. We also demonstrate that HDACi administration is functionally interfering with IRX1 and MLL-AF4, a finding which could help to improve new treatment options for t(4;11) patients.
Die Maillard-Reaktion findet während der Lagerung und thermischen Verarbeitung von Lebensmitteln zwischen den darin enthaltenen Proteinen und reduzierenden Kohlehydraten statt. Als Ergebnis der Reaktion entstehen sogenannte advanced glycation end products (AGEs), Protein-Derivate mit Glykierungs-Strukturen. Da Lebensmittel vor dem Verzehr häufig erhitzt werden, ist der Einfluss von AGEs auf die Pathogenese von Nahrungsmittelallergien von großem Interesse. Die Maillard-Reaktion könnte zur Bildung von neuen, für die Pathogenese der Nahrungsmittelallergie relevanten, Immunepitopen beitragen. Das Ziel dieser Arbeit war es, den Einfluss der Maillard-Reaktion auf die T-Zell-Immunogenität, die Antigenität und die von beiden Eigenschaften abhängige Allergenität von Nahrungsmittelallergenen zu untersuchen. Zunächst wurde der Einfluss der Maillard-Reaktion auf die T-Zell-Immunogenität von Ovalbumin (OVA), einem Allergen des Hühnereiweißes, untersucht. Dafür wurde glykiertes OVA (AGE-OVA) hergestellt indem das Protein zusammen mit Glukose erhitzt wurde. In dieser Arbeit konnte zum ersten Mal gezeigt werden, dass ein AGE-Derivat eines Lebensmittelallergens eine höhere T-Zellen-Immunogenität besitzt, als sein natives Gegenstück. Die Aktivierung und Proliferation von CD4+ T-Zellen durch AGE-OVA wurde in vitro durch Co-Kultivierung der T-Zellen mit dendritischen Zellen (DZ) untersucht. DZ sind professionelle Antigen- präsentierende Zellen, welche im Pathomechanismus der Allergie eine wichtige Rolle spielen. Im Vergleich zu nativen OVA und OVA welches ohne Glukose erhitzt wurde, führte die Stimulierung mit AGE-OVA zu einer deutlich erhöhten Aktivierung von OVA-spezifischen CD4+ T-Zellen. Damit DZ T-Zellen aktivieren können, muss das Allergen zunächst durch die DZ aufgenommen werden. In der vorliegenden Arbeit wurde gezeigt, dass die Aufnahme von AGE-OVA wesentlich höher war als die der Kontrollen. Außerdem konnte der scavenger receptor class A type I and II (SR-AI/II) als einer der hauptverantwortlichen Rezeptoren für die Aufnahme von AGE-OVA identifiziert werden. Zusammenfassend lässt sich aus den Ergebnissen dieser Arbeit die Hypothese aufstellen, dass die Glykierung von OVA eine erhöhte Assoziation des Allergens mit SR-AI/II ermöglicht, welche zu einer verstärkten Aufnahme des Allergens durch die DZ führt. Dadurch können mehr Peptide des Allergens an MHC II gebunden und auf der Zelloberfläche präsentiert werden. Das wiederum führt zur beobachteten stärkeren OVA-spezifischen CD4+ T-Zell-Aktivierung durch AGE-OVA. Als nächstes wurde die T-Zell-Immunogenität und Antigenität von AGE-OVA in vivo in einem Mausmodel untersucht. Es zeigte sich, dass AGE-OVA auch in vivo im Vergleich zu den nicht glykierten OVA-Formen eine erhöhte T-Zell-Immunogenität besitzt. Des weiteren führte die Immunisierung mit AGE-OVA zu einer erhöhten Produktion von IgE-Antikörpern. Somit wurde in dieser Arbeit gezeigt, dass AGE-OVA in vivo nicht nur eine erhöhte CD4+ T-Zell-Immunogenität besitzt, sondern auch eine höhere Antigenität hat als natives und ohne Glukose erhitztes OVA. Diese Ergebnisse harmonieren gut miteinander da CD4+ T-Zellen eine zentrale Rolle in der Aktivierung von B-Zellen und der IgE-Produktion durch selbige Zellen spielen. IgE-Antikörper besitzen eine essentielle Funktion beim Auslösen der klinischen Symptomatik der Allergie. Zusammenfassend lässt deshalb sagen, dass die Maillard-Reaktion die Allergenität von OVA erhöhen könnte. Zum Schluss wurden noch die immunstimulatorischen Eigenschaften des Erdnussallergens (AGE)-Ara h 2 untersucht. Da Erdnüsse häufig ernsthafte allergische Reaktionen hervorrufen und selten roh verzehrt werden, war es vom großen Interesse den Einfluss der Maillard-Reaktion auf Immunogenität und Antigenität von rekombinanten Ara h 2 (rAra h 2) zu untersuchen. Es zeigte sich, dass die Glykierung von rAra h 2 durch die Maillard-Reaktion die T-Zellen-Immunogenität, als auch die Antigenität des Allergens reduziert. Abschließend lässt sich sagen, dass die Maillard-Reaktion die allergenen Eigenschaften von Lebensmittelallergenen erheblich beeinflusst indem es die T-Zell-Immunogenität des Allergens verändert. Die Mechanismen welche die T-Zell-Immunogenität beeinflussen wurden hier näher untersucht. Wenn die Glykierung nicht die Bindung der T-Zellen- und/oder B-Zellen-Rezeptoren inhibiert, wird die Allergen-spezifische CD4+ T-Zell-Aktivierung und die davon abhängige IgE-Produktion dadurch erhöht, dass das glykierte Allergen durch DZ verstärkt über SR-AI/II aufgenommen wird. Die vorliegende Arbeit liefert wertvolle Information über die Allergenität von Proteinen die durch die Maillard-Reaktion modifiziert wurden and trägt dazu bei die Mechanismen von Nahrungsmittelallergien besser zu verstehen.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
Proteins and glycolipids have been found to be decorated with phosphorylcholine (PC) both in protozoa and nematodes that parasitize humans and animals. PC epitopes can provoke various effects on immune cells leading to an immunomodulation of the host’s immune system that allows long-term persistence of the parasites. So far, only a limited number of PC-modified proteins, mainly from nematodes, have been identified. Infections caused by Leishmania spp. (e.g., L. infantum in southern Europe) affect about 12 million people worldwide and are characterized by a wide spectrum of clinical forms in humans, ranging from cutaneous to fatal visceral leishmaniasis. To establish and maintain the infection, these protozoa are dependent on the secretion of effector molecules into the host for modulating their immune system. In this project, we analyzed the PC modification of L. infantum promastigotes by 2D-gel based proteomics. Western blot analysis with the PC-specific antibody TEPC-15 revealed one PC-substituted protein in this organism, identified as eEF1α. We could demonstrate that the binding of eEF1α to one of its downstream effectors is dependent on its PC-modification. In this study we provide evidence that in this parasite the modification of eEF1α with PC may be essential for its function as an important virulence factor.
The analysis of doxorubicin-loaded poly(butyl cyanoacrylate) nanoparticles in in vitro glioma models
(2005)
The use of doxorubicin for the treatment of glioma tumours would be an important approach in the chemotherapy treatment since doxorubicin is a very effective neoplastic agent. However, one problem faced by the use of doxorubicin for the treatment of brain tumours is the fact that doxorubicin is a substrate of an efflux pump protein, P-glycoprotein (P-gp), which is located on the luminal side of the brain capillary endothelium and in many tumour cells, which acts pumping out of the cell such substrate, and blocking its transport into the cell. A strategy to enhance the doxorubicin delivery into the brain would be the use of nanoparticles. This work showed, that the treatment of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles decreased the viability of the three glioma cell lines, the GS-9L, the RG-2, and the F-98 cell lines significantly in comparison to doxorubicin in solution, indicating an improvement of the nanoparticles-bound doxorubicin transport into the cells. The modification of the nanoparticles surface with different surfactants may even enhance the delivery of the drug into the cells. Searching for an improvement of the doxorubicin internalization, the nanoparticles surface was modified using polysorbate 80, poloxamer 188 and poloxamine 908 surfactants. The poloxamer 188 and polaxamine 908 surfactant modified nanoparticles did not show a significant enhancement of the doxorubicin internalization. Contrary, the treatment of polysorbate 80 surfactant modified nanoparticles led in some cases to a significant decrease of cancer cell viability. The use of doxorubicin in the three glioma cell lines allowed the measurement of different responses towards doxorubicin treatment. The different responses were due to the entry of various amounts of doxorubicin into the glioma cells, which express the P-glycoprotein in their cellular membrane. A higher level of the P-gp expression correlated with a weaker response towards the doxorubicin treatment. The GS-9L cell line showed a significant higher level of P-gp expression than the F-98, and RG-2 cell lines, and consequently, the GS-9L cell line presented the highest resistance to doxorubicin with the highest viability values after doxorubicin treatment. Due to the fact that the transport of doxorubicin is governed by the activity of the P-gp in the studied glioma cells, the use of poloxamer 185 as a P-gp inhibitor resulted in an enhancement of the uptake as well as of the accumulation of doxorubicin into the cells. The effect of poloxamer 185 on the doxorubicin uptake was significant marked in the case of doxorubicin-resistance cells, as the GS-9L cell line. In some cases, the presence of the nanoparticles formulation showed also an influence on such uptake improvement. The use of a P-gp inhibitor in combination with chemotherapeutic agents leads to encouraging results. Because of the wide spectrum of substances acting as P-gp inhibitors, the exact inhibitory mechanisms remain still unclear. For instance in our results the evaluation of a described P-gp inhibitor, polysorbate 80 did not show an important improvement in the doxorubicin uptake in the P-gp-expressing cell line, GS-9L. On the other hand, the Polysorbate 80-Dox-PBCA nanoparticles formulation decreased in greater extend the viability of the glioma cells than the poloxamer185-Dox-PBCA nanoparticles. Although, the P-gp inhibition was undoubtedly higher in the presence of poloxamer 185, polysorbate 80 showed a main effect on the disruption of the cellular membrane, resulting in an important cellular viability decrease. It seems that poloxamer 185 presents a direct effect on the functionality of the P-gp protein, which would be of great importance in the sensitization of resistant cancer cells. The range of concentration of poloxamer 185 is very important to yield an inhibitory effect on the P-gp-mediated transport mechanism. The accumulation of Rhodamine-123 (Rho-123), a known P-gp substrate, increased in a range of concentration from 0.001 % to 0.01, whereas at 0.1 % poloxamer 185 the accumulation significantly decreased. A maximal Rho-123 accumulation was reached at 0.01 % poloxamer 185.
A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI (Npr2fl/fl;Wnt1Cre and cGKIKO/fl;Wnt1Cre) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in Npr2fl/fl;Wnt1Cre mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli.
Carma-1 is required for B cell receptor-/CD40- and T cell receptor-/CD28-induced B- and T-cell activation via JNK and NF-betaB. In B cells, Carma-1 becomes phosphorylated by PKCbeta, leading to its oligomerization. Subsequent Bcl10 binding induces IKKbeta-activation and, thereby, canonical NF-KB signalling. Despite these findings it is still unknown how exactly Carma-1 is connected to the plasma membrane and to the IKK-complex. Therefore, we purified Carma-1 complexes from mouse CH12 B cells using anti-Carma-1 affinity columns. Mass spectrometric analyses of the column eluates demonstrated the presence of Carma-1 as well as three previously uncharacterized adaptor proteins in B cells, one of which was the Trk-fused gene (Tfg), an adaptor protein containing PB1 and coiledcoil domains. Whereas Tfg was originally identified as fusion partner of oncogenic Trk tyrosine kinase mutants, the normal cellular homologue of Tfg has so far not been described in B cells. However, Tfg has been shown in other systems to interact with IKKgamma and to enhance TNFinduced NF-KB activation. Tfg and Carma-1 co-localized at the plasma membrane and perinuclear structures in B cells. We further corroborated the interactions of Tfg, IKKgamma and Carma-1 by Blue Native gel electrophoresis, where Carma-1 and Tfg formed a 0.7–1 MDa complex. Ectopic expression of Tfg increased the molecular mass of IKKgamma complexes, fused IKKgamma, Bcl10 and Carma-1 complexes to a ~2 MDa complex, and increased basal and CD40-induced canonical activity of NF-KB and IKKbeta. In contrast, shRNA-mediated silencing of Tfg decreased CD40-induced IKKbeta activity. Very interestingly, in primary B cells, highest expression of Tfg was detected in marginal zone and B1 B cells, and Carma-1 and Tfg formed complexes in these B cells. Since Carma-1 is required for marginal zone B cell and B1 B cell development, we suggest that a functional interaction between Carma-1 and Tfg contributes to development and maintenance of these cells by means of canonical NF-KB signals.
Background. TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim. To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands.
Methods. Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy.
Results. CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model.
Conclusions. Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.
Objective: Establishment of an immunocompetent mouse model representing the typical progressive stages observed in malignant human gliomas for the in vivo evaluation of novel target-specific regimens.
Methods: Isolated clones from tumours that arose spontaneously in GFAP-v-src transgenic mice were used to develop a transplantable brain tumour model in syngeneic B6C3F1 mice. STAT3 protein was knocked down by infection of tumour cells with replication-defective lentivirus encoding STAT3-siRNA. Apoptosis is designed to be induced by soluble recombinant TRAIL + chemical Bcl-2/Bcl-xL inhibitors.
Results: Striatal implantation of 105 mouse tumour cells resulted in the robust development of microscopically (2 – 3 mm) infiltrating malignant gliomas. Immunohistochemically, the gliomas displayed the astroglial marker GFAP and the oncogenic form of STAT3 (Tyr-705-phosphorylated) which is found in many malignancies including gliomas. Phosphorylated STAT3 was particularly prominent in the nucleus but was also found at the plasma membrane of peripherally infiltrating glioma cells. To evaluate the role of STAT3 in tumour progression, we stably expressed siRNA against STAT3 in several murine glioma cell lines. The effect of STAT3 depletion on proliferation, invasion and survival will be first assessed in vitro and subsequently after transplantation in vivo. Upstream and downstream components of the STAT3 signalling pathway as well as possible non-specific side effects of STAT3-siRNA expression after lentiviral infection will be examined, too.
Conclusions: Its high rate of engraftment, its similarity to the malignant glioma of origin, and its rapid locally invasive growth should make this murine model useful in testing novel therapies for malignant gliomas.
Gene therapy is a promising therapeutic strategy that emerged from the attractive idea of targeting therapy at the molecular level. For many patients who suffer from genetic and acquired diseases that cannot be effectively treated by conventional treatment approaches gene therapy remains a huge hope of cure in spite of the hurdles regarding efficacy and safety that need to be overcome. The development of efficient gene transfer vehicles, mainly retroviral vectors, led to the first successful gene therapy trial, to treat patients suffering from X-linked severe combined immunodeficiency syndrome (X-SCID) using gene modified stem cells (Hacein-Bey-Abina, Le Deist et al. 2002). Despite the success of this trial, it revealed the danger of retroviral insertional mutagenesis as a major adverse event of gene therapy using gene-modified stem cells (Hacein-Bey-Abina, von Kalle et al. 2003). In contrast to stem cells, T cells are relatively resistant to insertional mutagenesis and transformation even after transduction with potent oncogenes using retroviral vectors (Newrzela, Cornils et al. 2008). However, mature T cells can self-renew, proliferate and survive for long periods. These criteria are supposed to render T cells prone to transformation. Therefore, the questions of mature T cells transformability and the control mechanism limiting their transformation are still elusive.
Chromosomal translocations of the human mixed-lineage leukemia (MLL) gene have been analyzed for more than 20 yr at the molecular level. So far, we have collected about 80 direct MLL fusions (MLL-X alleles) and about 120 reciprocal MLL fusions (X-MLL alleles). The reason for the higher amount of reciprocal MLL fusions is that the excess is caused by 3-way translocations with known direct fusion partners. This review is aiming to propose a solution for an obvious problem, namely why so many and completely different MLL fusion alleles are always leading to the same leukemia phenotypes (ALL, AML, or MLL). This review is aiming to explain the molecular consequences of MLL translocations, and secondly, the contribution of the different fusion partners. A new hypothesis will be posed that can be used for future research, aiming to find new avenues for the treatment of this particular leukemia entity.
A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor diflapolin was designed, synthesized, and characterized by 1H NMR, 13C NMR, and elemental analysis. These novel compounds were biologically evaluated for their inhibitory activity against sEH and FLAP. Molecular modeling tools were applied to analyze structure–activity relationships (SAR) on both targets. Results show that even small modifications on the lead compound diflapolin markedly influence the inhibitory potential, especially on FLAP, suggesting very narrow SAR.
The results presented here strongly indicate that ubiquitination of the recombinant human alpha1 GlyR at the plasma membrane of Xenopus oocytes is involved in receptor internalisation and degradation. Ubiquitination of the human alpha1 GlyR has been demonstrated by radio-iodination of plasma membrane-boundalpha1 GlyRs, whose subunits differed in molecular weight by additional 7, 14 or 21 kDa, corresponding to the molecular weights of one, two and three conjugated ubiquitin molecules, respectively, and by co-isolation of the non-tagged human alpha1 GlyR through hexahistidyl-tagged ubiquitin. Ubiquitin conjugated GlyRs where prominent at the plasma membrane, but could be hardly detected in total cell homogenates, indicating that ubiquitination takes place exclusively at the plasma membrane. Ubiquitination of the alpha1 GlyR at the plasma membrane was no longer detectable when the ten lysine residues of the cytoplasmic loop between transmembrane segments M3 and M4 were replaced by arginines. Despite this proteolytic cleavage continued to take place at the same extent as with the wild type alpha1 GlyR, suggesting that removal of GlyRs from the plasma membrane and routing to lysosomes for degradation were not dependent on ubiquitination. Also replacing a tyrosine in position 339, which was speculated to be part of an additional endocytosis motif, did not lead to a significant reduction of cleavage of the GlyR alpha1 subunits. However, a mutant lacking both, ubiquitination sites and 339Y, was significantly less processed. These results may suggest that the GlyR alpha1 subunit harbors at least two endocytosis motifs, which may act independently to regulate the density of alpha1 GlyR. Apparently, each of the two signals may be capable of compensating entirely the loss of the other. Part two of this Dissertation demonstrates that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment. Part three of this Dissertation describes my contribution (blue native PAGE analysis of metabolically labeled alpha7 and 5HT3A receptors and the examination of the glycosylation state of metabolically labeled alpha7 subunits) to a work on the limited assembly capacity of Xenopus oocytes for nicotinic alpha7 subunits. While 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors. Part four of this Dissertation describes my contribution (the biochemical analysis of the human P2X2 and P2X6 subtypes) to studies on the quaternary structure of P2X receptors. Armaz Aschrafi, the main author of the paper showed that subsequent to isolation under non-denaturing conditions from Xenopus oocytes the His-rP2X2 protein migrated on blue native PAGE predominantly in an aggregated form. The only discrete protein band detectable could be assigned to homotrimers of the His-rP2X2 subunit. Because of the exceptional assembly-behaviour of the rP2X2 protein compared to the rP2X1, rP2X3, rP2X4 and rP2X5 proteins, its human orthologue was investigated in the same manner. In contrast to rP2X2 subunits, hP2X2 subunits migrated under virtually identical conditions in a single defined assembly state, which could be clearly assigned to a trimer. P2X6 subunits represent the sole P2X subtype that is unable to form functional homomeric receptors in Xenopus oocytes. The blue native PAGE analysis of metabolically labeled hP2X6 receptors and the examination of the glycosylation state revealed that hP2X6 subunits form tetramers and aggregates that are not exported to the plasma membrane of Xenopus oocytes.
Die Beteiligung an Schlüsselfunktionen in zellulären Signalwegen macht Kinasen zu einem vielversprechenden Ansatzpunkt in der Wirkstoffentwicklung bei verschiedenen menschlichen Erkrankungen wie z.B. Krebs oder auch Autoimmun- und Entzündungskrankheiten. Die Prävention von post-translationalen Modifikationen durch Phosphorylierung und somit die Regulierung der nachgeschalteten Signalwege ist das Ziel von Kinaseinhibitoren. Die katalytische Aktivität von Kinasen ist abhängig von ATP, welches im hochkonservierten aktiven Zentrum bindet. Bedingt durch diese kinomweite hohe Konservierung stellt die Entwicklung von hoch selektiven ATP-mimetischen Inhibitoren eine Herausforderung dar. Typische ATP-Mimetika sind flach und die oft hydrophoben Moleküle weisen meist eine große Zahl an frei rotierbaren Bindungen auf. Um das aus dieser Flexibilität hervorgehende Problem der teils mangelnden Selektivität zu umgehen, kann eine bioaktive Konformation des Inhibitors durch Makrozyklisierung fixiert werden. Als Konsequenz dieser konformationellen Einschränkung können die entropischen Kosten während des Bindens reduziert werden und folglich zu einer gesteigerten Affinität gegenüber der Kinase führen.
Der Grundstein dieser Arbeit war der makrozyklische Pyrazolo[1,5-a]pyrimidin basierte FLT3 Kinaseinhibitor ODS2004070 (37). Im Rahmen eines kinomweiten Screenings konnten hohe Affinitäten zu verschiedensten Kinasen detektiert werden, was 37 zu einer guten Leitstruktur für das Design von potenten und selektiven Kinaseinhibitoren machte. Im Rahmen dieser Arbeit blieb das literaturbekannte Pyrazolo[1,5-a]pyrimidin basierte ATP-mimetische Bindemotiv sowie das makrozyklische Grundgerüst 37 bis auf einige wenige Variation unverändert.
Strukturelle Optimierungen zur Fokussierung der Selektivität wurden am sekundären Amin zwischen Bindemotiv und Linker als auch über die freie Carbonsäure durchgeführt. Mit einer Anzahl von mehr als 430 identifizierten Phosphorylierungsstellen ist die pleiotropisch und konstitutiv aktive Casein Kinase 2 (CK2) an verschiedensten zellulären Prozessen wie dem Verlauf des Zellzyklus, der Apoptose oder der Transkription regulatorisch beteiligt. Die Fehlregulation von CK2 wird häufig mit der Pathologie von Krankheiten wie zum Beispiel Krebs assoziiert, was CK2 zu einem vielversprechenden Ziel klinischer Untersuchungen macht.
Im Rahmen des CK2-Projekts war es möglich, durch spezifische Modifikationen an 37, die hoch selektiven und potenten CK2-Inhibitoren 47 und 60 zu entwickeln. Ebenfalls gezeigt wurde, dass kleine strukturelle Veränderungen, wie z.B. Makrozyklisierung, einen signifikanten Effekt auf Selektivität und Potenz des Inhibitors haben kann.
Weiter Untersuchungen der Verbindungen lenkten den Fokus weiterer Arbeiten u.a. auf die Serin/Threonin Kinase 17A (STK17A) oder auch death-associated protein kinase-related apoptosis-inducing protein kinase 1 (DRAK1) genannt. Sie ist Teil der DAPK Familie und gehört zusammen mit anderen Kinasen zu den weniger erforschten Kinasen. Bis heute ist nicht viel über ihre zellulären Funktionen und die Beteiligung an pathophysiologischen Prozessen bekannt. Berichtet wurde jedoch eine Überexpression in verschiedenen Formen von Hirntumoren des zentralen Nervensystems (Gliom). Strukturelle Modifikationen, unter Erhalt des makrozyklischen Grundgerüsts 37, führten zu dem hoch selektiven und potenten DRAK1 Inhibitor 121, der alle Kriterien für eine chemical probe Verbindung erfüllt.
Ein weiteres Ziel dieser Arbeit war die AP-2-assoziierte Protein Kinase 1 (AAK1) aus der NAK Familie, bestehend aus AAK1, BIKE und GAK. Sie ist als potenzielles therapeutisches Ziel für viele verschieden Krankheiten wie z.B. neuropathische Schmerzen, Schizophrenie und Parkinson identifiziert. Durch die Regulierung der Clathrin-mediierten Endozytose ist AAK1 an intrazellulären Bewegungen verschiedener nicht zusammenhängenden RNS- und DNSViren, wie beispielsweise HCV, DENV oder EBOV, beteiligt. Ebenfalls berichtet wurde eine mögliche Assoziation mit dem SARS-CoV-2 Virus, was das Interesse an neuen selektiven AAK1 Inhibitoren verstärkte. Die Entwicklung der hochpotenten und selektiven AAK1 Inhibitoren 61 und 63 basierte ebenfalls auf dem makrozyklischen Grundgerüst 37, das bereits im CK2- und DRAK1-Projekt verwendet wurde.
Zusammenfassend lässt sich sagen, dass es im Rahmen dieser Arbeit gelungen ist, ausgehend von einem höchst unselektiven makrozyklischen Grundgerüst, hochpotente und selektive Kinaseinhibitoren für CK2, DRAK1 und AAK1 zu entwickeln und zu charakterisieren. Im Zuge von Untersuchungen verschiedener Struktur-Wirkungsbeziehungen wurde gezeigt, dass es durch geringfügige strukturelle Modifikationen möglich ist, die kinomweite Selektivität zu variieren und auf eine Kinase zu fokussieren. Diese Arbeit brachte nicht nur die erwähnten Inhibitoren hervor, sondern bildet auch die Grundlage für weitere Projekte zur Entwicklung von hoch potenten und selektiven Verbindungen als potenzielle chemische Werkzeuge für den Einsatz in der Forschung.
Lipid mediators have been referred as bioactive lipids, whose change in lipid levels resulted in functional or pathophysiological consequences. They are in the focus of biological research, nevertheless this is a late recognition due to the many difficulties of working with bioactive lipids due to their properties: hydrophobic, unstable and they occur in only in small quantities. Liquid chromatography and mass spectrometry have facilitated the work with them. Especially in this field, cardiovascular diseases and inflammatory mediated diseases and cancer are pathophysiological events where LMs are deregulated. Additionally, if the modulation of one LM pathway is not sufficient to overcome a disease, the combination of targeting two or more pathways could be effective. Needless to say, lipid signaling cascades are complicated pathways and possible shunting into other pathways when inhibiting or genetically deleting enzymes should be taken into consideration.
The first part of this work has focused on enzymes that metabolize eicosanoids, like mPGES-1 and 5-LO. mPGES-1 is an important enzyme metabolizing PGH2 and one of the key players of the AA cascade. Its product, PGE2 plays an important role in different inflammatory processes. Inhibition of the mPGES-1 might be a promising step to circumvent COX dependent side effects of NSAIDs. The class of quinazoline compounds around the lead structure FR20 has been investigated on isolated human and murine enzyme, in HeLa cells and in different human whole blood (HWB) settings to establish the possible effects of these compounds on eicosanoid profiling. Novel compounds with inhibitory activities in the submicromolar range (IC50: 0.13 µM - 0.37 µM on isolated enzyme) were obtained which were also effective in cells and HWB. Furthermore, pharmacological profiling of toxicity and lipid screening with LC/MS-MS revealed that compounds also reduce PGE2 levels in intact cells and whole blood; they do not impair cell viability but lack the ability to inhibit the murine mPGES-1 enzyme. This problem could be overcome by means of chemical synthesis varying the scaffold (quinoline, quinazoline) or introducing biosteric replacement in the phenyl moieties.
5-LO is a relevant enzyme that plays an important role in eicosanoid signaling in particular in leukotriene biosynthesis. Leukotrienes are involved in asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, sepsis, cancer and atherosclerosis. Moreover, genetic variants in the genes of the 5-LO pathway have been associated with the risk of development of acute myocardial infarction and stroke. Eicosanoids are increased in infectious exacerbations of chronic obstructive pulmonary disease (COPD). They are also elevated in the airways of stable COPD patients compared to healthy subjects. Therefore, 5-LO has attired the scientific community as a possible therapeutic target to treat the several disease conditions listed before. In this study an extensive evaluation of imidazo[1,2-a]pyridines as a suitable lead structure for novel 5-LO targeting compounds was presented Within the three publications, 5-LO inhibitory activity of synthesized compounds was investigated in intact PMNL, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and compounds were in vitro pharmacological evaluated. Chemical modifications for lead optimization via straight forward synthesis were used to combine small polar groups (hydroxy, and methoxy groups) which led to a suitable candidate with desired in vitro pharmacokinetic profile in terms of solubility and intrinsic clearance without showing any cytotoxicity. More than 70 imidazo[1,2-a]pyridine derivatives have been synthesized, resulting in more than 50 active compounds. Although it was not possible to introduce a solubility group without impairing the 5-LO inhibitory activity, combination of small polar groups lead to a more favorable solubility and in vitro metabolic stability. Overall, the development of 5-LO inhibitors with high efficacy and selectivity in vivo will provide a possible treatment for patients having one of the diseases where leukotriene biosynthesis plays an important role.
Other types of 5-LO inhibitors have been synthesized during this work, NO-NSAIDs can be postulated as novel 5-LO inhibitors that could circumvent the undesired side-effects of inhibiting COX isoforms (ulcer perforation, gastrointestinal bleeding and in some cases death). It is suggested that NO group is released in situ or after compounds are metabolized. NO-NSAIDs maintain the same anti-inflammatory properties by inhibiting 5-LO in clinical relevant concentrations. NO-NSAIDs are currently under clinical trial for the treatment of diseases where inflammation plays an important role. Synthesis of NO-NSAIDs is straightforward and can be applied for most NSAIDs recently published. Among them, the most promising candidate is NO-sulindac that was able to inhibit 5-LO product formation in intact PMNL, purified 5-LO and HWB in micromolar concentration. Additional experiments regarding their mechanism are currently being performed.
The present study could show that dual inhibitors are an interesting approach that is practicable. It has been used in the recent years to overcome side-effects and diseases concerning more pathophysiological conditions. MetS is an example of a conjunction of symptoms: hyperglycemia, hypertriglyceridemia, hypertension and obesity. Due to its complex nature, the current treatment strategies of MetS require multiple pharmacological compounds regulating lipid and glucose homeostasis as well as blood pressure and coagulation. This study describes the first synthesis of dual sEH/PPAR modulators as potential agents for treatment of MetS. Following a combinatorial approach, an acidic head group known as a pharmacophore important for PPARα/γ dual agonistic activity was combined with different hydrophobic urea derivatives in order to introduce an epoxide mimetic (sEH pharmacophore). The resulting compounds yielded high inhibition of sEH and different patterns of PPAR agonistic activity. This study demonstrates that the pharmacophores of PPAR agonists and sEH inhibitors can be easily combined, resulting in a simplified blueprint of a dual sEH/PPAR modulator. Further in vivo pharmacological evaluation studies are needed in order to evaluate, which pattern of PPAR activation shows the most promising profile for treatment of metabolic syndrome.
Another example of dual pharmacology has been presented in this work. Natural products derived compounds were able to target sEH and exhibit promising antiproliferative properties. The principle of addressing multiple targets by natural products can be transferred to synthetic multi-target ligands. In conclusion, several (E)-styryl-1H-benzo[d]imidazoles were synthesized and evaluated on recombinant sEH after an initial hit (IPS) that lead to potent sEH inhibitors exhibiting antiproliferative activities. Following the natural product-inspired design, the desired biological activity from a bacterial secondary metabolite has been enhanced and transferred to a synthetic compound series. The resulting compounds were accessible via an easy synthetic route and offered a possibility to investigate the structure-activity relationships. The natural product inspired drug design extends the valuable role of natural products as drugs and drug precursors to templates for fully synthetic bioactive molecules. Simplification of natural products by means of chemical synthesis could lead to an interesting field in the treatment of cancer.
Affinity chromatography has been used to unravel unknown- and off-target effects which either contribute to the biological effect of the inhibitor or that counteract or lead to undesired side-effects. During this PhD work, two main projects related to this technique have been established. In the first one, related to an imidazo[1,2-a]pyridine inhibitor (EP6), it has been shown that epoxide-sepharose is a reliable material in order to couple compounds bearing an alcohol. Coupling of an analogue of EP6 to the sepharose has been accomplished and affinity towards 5-LO was demonstrated. The challenging step is to discern from unspecific protein binders and analysis via SDS-PAGE separation and mass spectrometry. Further experiments using other cell types or improving SDS-PAGE analysis (e.g. 2D gel analysis) should be useful to unravel EP6 off-target effect. During the second project related to off-target effects of celecoxib and DMC, the main problem was the coupling of the functional group to the sepharose. Affinity towards COX-2 could not be demonstrated pointing out the inefficient coupling method. Higher pH values during coupling reaction should be tested in further experiments. Nevertheless, affinity chromatography is a useful technique to unravel cellular mechanisms.
Sphingolipid metabolism is also a recent area that attired the attention of cancer researchers, due to their important roles in cell proliferation and apoptosis. Ceramide metabolism inhibitors were synthesized and evaluated on different assay systems in order to assess their efficacy on several cancer lines. Remarkably, 2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (32) was a useful scaffold to mimic the sphingoid base. This key intermediate was used to produce ceramide analogues that could enter the cell and target apoptosis machinery. EB143 (38) increased ceramide levels in an in vitro ceramide synthase assay in a dose-response manner meaning that ceramide synthase was not inhibited but the ceramide de novo synthesis was activated. This effect was due to the fact that EB143 is a cytotoxic compound with an interesting antiproliferative profile. Further chemical modifications should be carried out to modulate this effect.
COX and LO inhibitors are cancer-preventive not only by inhibiting specific antiapoptotic AA metabolites but also by facilitating accumulation of AA which promotes neutral SMase activity and increases the proapoptotic ceramide. Several 5-LO inhibitors have been evaluated on several cancer lines and sphingolipid levels were measured in order to obtain a relationship. A549, Capan-2 and MCF-7 cells line were incubated with synthetic 5-LO inhibitors and zileuton. Compounds were cytotoxic to all cancer cell lines except from A549. Needless to say, zileuton did not exhibit a cytotoxic profile. Synthetic 5-LO inhibitors were able to modify ceramide levels but were useless when coincubating with sphingolipid metabolism inhibitors (myoricin, amitryptiline etc.) and inconsistent results were obtained. On the contrary, zileuton selectively increased Cer-C16 levels and in less extend Cer-C24:1. When using a SPT inhibitor (myoricin) alone was able to reduce C24:1 and Cer-C16:0 levels below the control, a similar effect occurred when incubation the cells with zileuton and myriocin. Interestingly, treatment of zileuton together with either amitryptiline or desipramine led to a decrease in Cer-C24:1 and levels Cer-C16:0 but the inhibition was not complete indicating that probably the de novo pathway has an important role. Further investigations on mRNA level should be carried out in order to discern which CerS is activated.
The main objective of the present thesis was the synthesis of lipid signaling modulators and their evaluation in vitro as therapeutic strategy to overcome pathophysiological conditions (cancer, metabolic syndrome, etc). It has been accomplished on many relevant targets like 5-LO, mPGES-1, sEH and PPAR and these lipid signaling modulators could be used in the treatment of diseases conditions where lipid mediators play an important role.
IL-18, a recently identified member of IL-1 family, is now recognized as an important regulator of innate and acquired immune responses. Therefore, the antitumor activities of IL-18 have been investigated. IL-18 has been shown to induce IFN-γ production by T, B, and NK cells, enhances NK cell activity, activates Fas ligandmediated apoptosis of the tumor cells, and improves the overall antitumor immunity. KG-1 cells were derived from a patient with acute myeloid leukemia (AML). IL-18 has been shown to induce IFN-γ production in those leukemic cells. TLR-3, in addition to its ability to recognize viral double stranded RNA, also can recognize the synthetic analogue poly(I:C) and induces type I IFN, inflammatory cytokine production, e.g TNF-α, and maturation of denderitic cells. In the present work the potential modulatory effect of PIC on IFN-γ and TNF-α production by KG-1 cells treated with IL-18 was investigated. Indeed, PIC strongly amplified the production of IFN-γ induced by IL-18 on mRNA and protein levels via NF-κB as well as p38 and JNK MAPK activation. Compared to IFN-γ, TNF-α showed different behaviour in KG-1 cells. On mRNA level I found only weak induction of TNF-α by IL-18 which was potentiated in the presence of PIC. Similarly, the release of TNF-α by IL-18 plus PIC required NF-κB as well as p38 and JNK MAPK activation. Furthermore, in the present work I found that TLR-3 is required for IFN-γ and TNF-α production. In addition, it is demonstrated by immunofluoresence that TLR-3 is localized in cytoplasm but not on the cell surface in KG-1 cells. Recently, it has been demonstrated that IFN-γ shows therapeutic potential as detected in AML blasts, specifically via inhibition of proliferation and induction of apoptosis. Thus our data could serve as a rationale for the clinical use of PIC and IL-18 in combination therapy. In search for new cytokines potentially modulated by the combination IL-18 plus PIC in KG-1 cells, cytokine antibody array analysis was performed. I found an upregulation of expected genes like IP-10 but most interestingly unexpected upregulation of PDGF-AA. Searching for detailed mechanisms of PDGF-AA induction, I found that neither p38 nor JNK is involved in PDGF-AA production but NF-κB is essential for the expression of PDGF-AA. Furthermore, I found that PDGF-AA is not able to increase the proliferation of KG-1 cells. PDGF and TGF-β are examples of signaling molecules which control the growth, survival, motility, and differentiation of cells. Therefore, the release of TGF-β by IL-18 plus PIC was monitored by ELISA. The level of TGF-β in cellular supernatants revealed that neither PIC nor IL-18 was able to significantly mediate release of TGF-β indicating that only PDGF-AA but not TGF-β is induced by PIC and IL-18 in KG-1 cells. To the best of our knowledge this is the first time that IL-18 or PIC is shown to induce the expression of PDGF-AA in KG-1 cells.
Nerve tissue contains a high density of chemical synapses, about 1 per µm3 in the mammalian cerebral cortex. Thus, even for small blocks of nerve tissue, dense connectomic mapping requires the identification of millions to billions of synapses. While the focus of connectomic data analysis has been on neurite reconstruction, synapse detection becomes limiting when datasets grow in size and dense mapping is required. Here, we report SynEM, a method for automated detection of synapses from conventionally en-bloc stained 3D electron microscopy image stacks. The approach is based on a segmentation of the image data and focuses on classifying borders between neuronal processes as synaptic or non-synaptic. SynEM yields 97% precision and recall in binary cortical connectomes with no user interaction. It scales to large volumes of cortical neuropil, plausibly even whole-brain datasets. SynEM removes the burden of manual synapse annotation for large densely mapped connectomes.
Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARalpha and PLZF/RARalpha or AML-1/ETO activate Wnt signaling by upregulating gamma-catenin and beta-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARalpha-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both beta-catenin and gamma-catenin in X-RARalpha-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARalpha-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARalpha, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.
In addition to infectious viral particles, hepatitis B virus-replicating cells secrete high amounts of SVPs, which are ssembled by HBsAg in the shape of spheres and filaments but lack any capsid and genome. Filaments are characterized by a much higher amount of the surface protein LHBs as compared to spheres. Spheres are
released via the constitutive secretory pathway, while viral particles are ESCRT-dependently released via MVBs. The interaction of virions with the ESCRT machinery is mediated by α-taxilin that connects the PreS1 domain of LHBs with the ESCRT-component tsg101. Since viral particles and filaments contain a significant amount of LHBs, it is unclear whether filaments are secreted as spheres or released like viral particles. To study the release pathways of HBV filaments in the absence of viral particles, A core-deficient
HBV mutant (1.2×HBVΔCore) was generated by site-directed mutagenesis based on wt1.2x HBV. The start codon of core protein was mutated into stop codon, which was confirmed by DNA sequencing. Data from HBsAg ELISA, Western blot, immunofluorescence microscopy and immunoelectron microscopy showed that the lack of core protein did neither affect the production nor the secretion of HBV SVPs. The intracellular distribution of
LHBs and SHBs showed no difference between wtHBV and the core-deficient mutant expressing cells. Therefore, this system is suitable to investigate the release pathway of HBV filaments in the absence of viral particles. Confocal microscopy analysis of cells cotransfected core-deficient mutants with peYFPRab7 as marker for the endosomal/MVB pathway or with pGalT-eGFP as marker for the trans Golgi apparatus showed that YFP-Rab7, but not GalT-GFP, partially colocalized with LHBs. Furthermore, LHBs could be found in dilated MVBs by immune electron microscopy of ultrathin sections. This was confirmed by isolation of MVBs by cell fractionation using discontinuous sucrose gradient ultracentrifugation and percoll-based linear gradient ultracentrifugation, indicating that filaments enter MVBs in the absence of virion formation. Moreover, inhibition of MVB biogenesis by the small molecular inhibitor U18666A significantly abolished the release of filaments in a dose-dependent manner, but no inhibition could be observed in the production. In contrast, no inhibition on the secretion and production of spheres could be
detected. Inhibition of ESCRT-functionality by coexpression of transdominant negative mutants (Vps4A, Vps4B, CHMP3) abolished the release of filaments while secretion of spheres was not affected. These data indicate that in contrast Abstract 73 to spheres while are secreted via the secretory pathway, filaments are released via ESCRT/MVB pathway like infectious viral particles.
Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2μM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport.
The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR),[1, 2] a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors.
Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation and accumulation of immature myeloblasts, which impair normal hematopoiesis. While this definition categorizes the disease into a distinctive group, the large number of different genetic and epigenetic alterations actually suggests that AML is not a single disease, but a plethora of malignancies. Still, most AML patients are not treated with targeted medication but rather by uniform approaches such as chemotherapy. The identification of novel treatment options likely requires the identification of cancer cell vulnerabilities that take into account the different genetic and epigenetic make-up of the individual tumors. Here we show that STK3 depletion by knock-down, knock-out or chemical inhibition results in apoptotic cells death in some but not all AML cell lines and primary cells tested. This effect is mediated by a premature activation of cyclin dependent kinase 1 (CDK1) in presence of elevated cyclin B1 levels. The anti-leukemic effects seen in both bulk and progenitor AML cells suggests that STK3 might be a promising target in a subset of AML patients.
Binding free energy calculations that make use of alchemical pathways are becoming increasingly feasible thanks to advances in hardware and algorithms. Although relative binding free energy (RBFE) calculations are starting to find widespread use, absolute binding free energy (ABFE) calculations are still being explored mainly in academic settings due to the high computational requirements and still uncertain predictive value. However, in some drug design scenarios, RBFE calculations are not applicable and ABFE calculations could provide an alternative. Computationally cheaper end-point calculations in implicit solvent, such as molecular mechanics Poisson–Boltzmann surface area (MMPBSA) calculations, could too be used if one is primarily interested in a relative ranking of affinities. Here, we compare MMPBSA calculations to previously performed absolute alchemical free energy calculations in their ability to correlate with experimental binding free energies for three sets of bromodomain–inhibitor pairs. Different MMPBSA approaches have been considered, including a standard single-trajectory protocol, a protocol that includes a binding entropy estimate, and protocols that take into account the ligand hydration shell. Despite the improvements observed with the latter two MMPBSA approaches, ABFE calculations were found to be overall superior in obtaining correlation with experimental affinities for the test cases considered. A difference in weighted average Pearson () and Spearman () correlations of 0.25 and 0.31 was observed when using a standard single-trajectory MMPBSA setup ( = 0.64 and = 0.66 for ABFE; = 0.39 and = 0.35 for MMPBSA). The best performing MMPBSA protocols returned weighted average Pearson and Spearman correlations that were about 0.1 inferior to ABFE calculations: = 0.55 and = 0.56 when including an entropy estimate, and = 0.53 and = 0.55 when including explicit water molecules. Overall, the study suggests that ABFE calculations are indeed the more accurate approach, yet there is also value in MMPBSA calculations considering the lower compute requirements, and if agreement to experimental affinities in absolute terms is not of interest. Moreover, for the specific protein–ligand systems considered in this study, we find that including an explicit ligand hydration shell or a binding entropy estimate in the MMPBSA calculations resulted in significant performance improvements at a negligible computational cost.