Previous publications from this laboratory demonstrated that administration of leukemia inhibitory factor (LIF) (125 µg/kg) to young, male Sprague-Dawley rats at 6, 24, and 48 h after middle cerebral artery occlusion (MCAO) reduced infract volume, improved sensimotor skills, and alleviated damage to white matter at 72 h after the injury. In vitro studies using cultured oligodendrocytes (OLs) showed that LIF (200 ng/ml) also protects against 24 h of oxygen-glucose deprivation through activation of Akt signaling and upregulation of the antioxidant enzymes peroxiredoxin IV and metallothionein III. Other groups have demonstrated that LIF reduces neurodegeneration in animal models of disease, but the neuroprotective mechanisms of LIF during permanent ischemia have not yet been examined. The overall hypothesis to be tested in this project is whether LIF exerts similar protective mechanisms against neurons during ischemia through increased antioxidant enzyme expression in neurons. In the first set of experiments, superoxide dismutase (SOD) activity was significantly increased in the ipsilateral hemisphere of LIF-treated rats compared to rats that received PBS treatment at 72 h after MCAO. Western blot and immunohistochemical analysis revealed that SOD3 was upregulated in brain tissue and induced specifically in cortical neurons tissue at this time point. Neurons that expressed high levels of SOD3 at 72 h after MCAO also showed high levels of phosphorylated Akt (Ser473). LIF (200 ng/ml) reduced necrotic and apoptotic cell death against 24 h of OGD as measured by lactate dehydrogenase (LDH) release and caspase-3 activation. Quantitative real-time PCR analysis showed that LIF treatment upregulated SOD3 gene expression in vitro during OGD. Treatment with 10 µM Akt Inhibitor IV and transfection with SOD3 siRNA counteracted the neuroprotective effects of LIF in vitro, showing that upregulation of SOD3 and activation of Akt signaling are necessary for LIF-mediated neuroprotection. Several transcription factors that regulated Akt-inducible genes were previously identified by this lab, including myeloid zinc finger-1 (MZF-1) and specificity protein-1 (Sp1). The goal of the second set of experiments was to determine whether LIF exerted protective actions through MZF-1 and Sp1. According to analysis with Genomatix, MZF-1 and Sp1 have multiple binding sites in the promoter for the rat SOD3 gene. Western blot analysis showed that there was a trend towards increased MZF-1 protein expression in the brains of LIF-treated rats that approached significance. Immunohistochemical analysis and quantitative real-time PCR showed a significant in vitro upregulation in MZF-1 expression among LIF-treated neurons compared to PBS-treated neurons. Sp1 gene expression was not changed by LIF treatment, but there was a trend towards increased protein expression. In addition, there was a significant correlation between Sp1 and MZF-1 among brain samples from LIF-treated rats but not PBS-treated or sham rats at 72 h after MCAO. Immunohistochemical analysis revealed that Sp1 and MZF-1 co-localized with neuronal nuclei and SOD3 at 72 h after MCAO. Neurons that were transfected with MZF-1 or Sp1 siRNA following isolation did not show a significant decrease in LDH release after 24 h OGD that was observed among neurons transfected with scrambled siRNA. These data demonstrate that Sp1 and MZF-1 are involved with the neuroprotective signaling of LIF under ischemia. This laboratory has demonstrated that LIF activates transcription of protective genes and increases the activity of transcription factors through modulation of intracellular signaling. However, the upstream signaling mechanisms of LIF during ischemia had not previously been investigated. Previous investigators found that the LIF-specific subunit of the heterodimeric LIF receptor (LIFR) is induced by CNS injury. Western blot analysis was used to determine whether LIFR was induced in the brain and the spleen, which plays a role in the peripheral immune response, after MCAO. According to these results, LIF treatment significantly upregulates LIF in the brain compared to PBS treatment or sham injury at 72 h after MCAO. Genomatix analysis of the LIFR promoter region revealed a binding site for Sp1, which is one of the transcription factors responsible for neuroprotection by LIF. At this same time point, splenic LIFR expression is significantly reduced after MCAO compared to sham injury. LIF treatment did not significantly increase LIFR expression, but did significantly increase spleen size compared to PBS treatment at 72 h after MCAO. Although there was a trend towards increased LIFR expression in the spleen from 24 h to 72 h after MCAO, this increase was not statistically significant. However, there was a significant positive correlation between spleen weight and LIFR expression among rats euthanized 24-72 h after MCAO/sham injury. In addition, there was a significant negative correlation between LIFR expression in the brain and the spleen weight, thus showing that LIFR is upregulated following the splenic response. According to findings from other groups, JAK1 has been shown to associate with the heterodimeric LIF receptor (LIFR/gp130) and directly activate PI3K/Akt signaling. To test whether JAK1 contributes neuroprotection during ischemia, cultured neurons were treated with several concentrations (2.5-50 nM) of GLPG0634, a JAK1-specific inhibitor prior to 24 h of OGD. With the exception of the 2.5 nM concentration, all concentrations of GLPG0634 significantly decreased LDH release compared to DMSO treatment, with the 5 nM concentration having the most potent effect on reducing cytotoxicity. However, the 5 nM concentration had no significant did not significantly reduce LDH release compared to DMSO treatment under 24 h of normoxic conditions. These results indicate that JAK1 activity is primarily detrimental to neurons during ischemia. Although it is possible that LIF signaling activates JAK1, it is unlikely that JAK1 is responsible for LIF-mediated neuroprotection during ischemia. The results of these experiments allowed us to determine several molecular mechanisms for LIF-mediated neuroprotection. LIF, which binds to its heterodimeric receptor, activates Akt signaling during ischemia. The transcription factors Sp1 and MZF-1, which are located downstream of Akt, bind to the promoter of the SOD3 gene. In addition, Sp1 also regulates the LIFR gene. SOD3 upregulation increases total SOD activity, which decreases apoptotic and necrotic cell death during apoptosis. Due to its ability to promote antioxidant expression and survival signaling in multiple neural cell types, LIF shows promise as a novel treatment for permanent focal cerebral ischemia.

Pour faciliter l’initiation de la transcription par l’ARN Polymérase II, le complexe co-activateur de la transcription SAGA possède une activité d’acétylation des histones H3 et une activité de déubiquitination des histones H2B, catalysée chez l’homme par l’enzyme USP22. Mon travail de thèse a porté sur l’étude de la régulation de cette activité de déubiquitination.Au sein de SAGA, USP22 interagit fortement avec trois protéines pour former un module structural appelé module de déubiquitination (DUBm). Nous avons montré que la formation d’un tel module était requise pour activer USP22. D’autre part, deux sous-unités du DUBm humain, ATXN7 et ATXN7L3, contiennent un domaine SCA7. Nos résultats montrent que le repliement structural adopté par ces deux doigts de zinc n’avait pas encore été décrit. Nous avons démontré que le domaine SCA7 de ATXN7 peut interagir avec un nucléosome in vitro et que cette interaction participe à la régulation fine de l’activité de déubiquitination de SAGA. Nous proposons qu’en interagissant avec le nucléosome, le domaine SCA7 de Sgf73 ou de ATXN7 pourrait positionner le DUBm de façon optimale par rapport à son substrat. / The SAGA complex is one of the most studied transcriptional co-activator complexes. To facilitate transcription by RNA Polymerase II, SAGA presents a modular organization and harbours two enzymatic activities. In human cells, these two enzymes are called GCN5 and USP22 and they can respectivelly acetylate histones H3 and deubiquitinate histones H2B. During my PhD thesis, I have worked on the regulation of SAGA deubiquitination activity. In the SAGA complex, USP22 interacts strongly with three other subunits to form a structural and functionnal module, named deubiquitination module (DUBm). We have shown that the free recombinant USP22 enzyme is not active, but that the formation of a stable DUBm triggers a strong stimulation of USP22 catalytic activity. Secondly, in human cells, two subunits of the DUBm, ATXN7 and ATXN7L3, contain a domain, called SCA7, that is not found in any other protein. Our results show that the new structural fold adopted by these two domains is specific to these zinc-fingers. These two SCA7 domains share a common structural heart, but their atomic structures reveal also differences, especially in the spatial organization of secondary structure elements. Indeed, we have shown that ATXN7 SCA7 domain can interact in vitro with a nucleosome which is not the case of ATXN7L3 SCA7 domain. Finally, I could show that in vivo the SCA7 domain of Sgf73, the ortholog of ATXN7 has a role in fine tunning SAGA deubiquitination activity. We hypothesize that the interaction between a nucleosome and the SCA7 domain of ATXN7 or Sgf73 would regulate SAGA deubiquitination activity by an optimal positionning of the module to its substrate.

Les transposases sont des enzymes qui reconnaissent des séquences spécifiques sur l'ADN aux bornes des transposons (éléments à transposer) et catalysent des réactions de coupure et de transfert de brins. La mobilité des transposons entraîne la plasticité des génomes, et dans certains cas, l'exaptation de gènes de transposons contribue à l'émergence de nouvelles fonctions cellulaires. La paramécie est un modèle eucaryote unicellulaire extraordinaire pour étudier le rôle des transposases domestiquées de PiggyBac (nommées PiggyMac et PiggyMac-like) dans le réarrangement programmé de son génome. Ces dernières contribuent à l'assemblage de son génome somatique au cours du cycle sexuel. Les principaux axes de ce projet se sont centrés sur l’étude structurale de la transposase PiggyBac (issue de Trichoplusia ni) et une étude d’interaction avec des séquences particulières de son transposon. Ainsi qu’une étude structurale de la transposase domestiquée PiggyMac chez la paramécie.La première structure obtenue (domaine riche en cystéine de la transposase PiggyBac) montre une structuration en doigt de zinc de type PHD-RING. Il a été démontré in vivo et in vitro l’importance du domaine riche en cystéines (CRD) de PiggyBac pour une activité d’excision et d’intégration du transposon PiggyBac. Nous avons pu mettre en évidence que le CRD de PiggyBac cible des séquences spécifiques d’ADN qui sont localisées dans les séquences TIR (Terminal Inverted Repeat) gauche et droite du transposon. Grâce aux résultats issus de la RMN, des modèles de complexes protéine-ADN ont pu être établis.Concernant PiggyMac (transposase PiggyBac domestiquée), son domaine riche en cystéines et histidines a pu être produit doublement marqué (15N et 13C) dans E.coli. Des études structurales en RMN et l'utilisation d'un programme de modélisation moléculaire CYANA ont permis d’accéder à la structure tridimensionnelle de ce domaine.Nous montrons que celui-ci se replie en doigt de zinc entrelacé qui lie deux ions zinc avec un total de huit histidines et cystéines (résultat en cours de publication). La configuration de ce doigt de zinc est différente de celui de PiggyBac et même nouveau dans la littérature concernant ce peptide. Cette étude a permis de mettre en évidence un nouveau type de doigt de zinc. / Transposases are enzymes that recognize specific DNA sequences across transposons (elements to transpose) and catalyze cleavage and transfer reactions strands. The mobility of transposons causes the genome plasticity, and in some cases, the transposon gene exaptation contributes to the emergence of new cellular functions. Paramecium is an extraordinary unicellular eukaryotic model to study the role of transposases domesticated PiggyBac (named PiggyMac and PiggyMac-like) in the programmed rearrangement of its genome. These contribute to the assembly of the somatic genome during sexual cycle. The main axes of this project have focused on the structural study of the transposase PiggyBac (derived from Trichoplusia ni) and an interaction study with particular sequences of the transposon. And a structural study of the transposase domesticated PiggyMac in Paramecium.The first structure obtained (cysteine-rich domain of the transposase PiggyBac) shows a structure in PHD-RING-type zinc finger. It has been demonstrated in vivo and in vitro the importance of the cysteine-rich domain (CRD) of PiggyBac for excision activity and integration of the transposon PiggyBac. We were able to show that the CRD PiggyBac target specific DNA sequences that are located in the TIR sequences (Inverted Terminal Repeat) left and right of the transposon. Thanks to the NMR results, protein-DNA complexes models were established.Regarding PiggyMac (PiggyBac domesticated transposase), its cysteine ​​and histidine rich domain has been doubly labeled product (15N and 13C) in E. coli. NMR structural studies and the use of a CYANA molecular modeling program allowed to access the three-dimensional structure of this domain.We show that it folds into interlaced zinc finger that binds two zinc ions with a total of eight histidine and cysteine ​​(results being published). The configuration of this zinc finger is different from that of PiggyBac and even new in the literature concerning this peptide. The study highlighted a new type of zinc finger.

This paper describes our continuing studies on stabilization of peptide structures in supersecondary conformations that are designed to mimic conformational antigenic epitopes. In this work we have used the consensus Cys2His2 zinc-finger peptide motif as a template to engineer and synthesize antigenic loop peptide segments from two protein antigens, lactate dehydrogenase C4 isozyme (LDH-C4) and human chorionic gonadotropin β subunit (β-hCG). Confirmation that the engineered peptide constructs assumed a zinc-finger conformation was obtained by absorption spectroscopy of the Co2+ complexes. The circular dichroism (CD) spectra of the free peptides show random coil conformations, while the Zn2+-complexed peptides acquired the zinc-finger motif upon titration with Zn2+, as evidenced by the appearance of absorbances indicating α-helix and some β-conformation. No peptide aggregation was observed, as these peptides were monomeric under all conditions tested. In order to examine the immunogenicity of the zinc-finger constructs, one sequence from LDH-C4 (ZFLMVF) and two sequences from β-hCG (ZF2TT3 and ZF4TT3) were selected and chimeras were synthesized to incorporate promiscuous T-cell epitopes from either tetanus toxoid or measles virus. The ZFLMVF construct was highly immunogenic in rabbits, and the ZF2TT3 and ZF4TT3 peptides were highly immunogenic in both mice and rabbits, eliciting high-titer antipeptide antibodies specific for their immunogenic sequences. However, the antibodies raised to the zinc-finger constructs showed minimal reactivity against their respective native protein antigens as determined by ELISA. This is surprising in the case of β-hCG, since the ZF2 zinc-finger peptide was an effective inhibitor of binding of anti-β-hCG-loop(38-57) antibodies to whole hCG, as assessed by a competitive inhibition radioimmunoassay. This implies that, although the cyclized 40-52 sequence from βhCG and the zinc-finger peptide ZF2 exhibit similar conformations in solution, the zinc-finger engineered loop is apparently not in a sufficiently correct conformation for antibody recognition of native hCG. Our results with the LDH-C4 zinc finger loop imply that antibody recognition of antigen involves specific side-chain interactions that must be maintained by a precise conformation.

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Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.

Methoden zur direkten Detektion oder Anreicherung von doppelsträngiger DNA (dsDNA) bieten ein hohes Potential zum Einsatz in der molekularen Diagnostik. Bereits etablierte Methoden für die Nukleinsäure - Detektion (NAD) basieren in der Regel auf der Hybridisierung des komplementären Stranges gefolgt von der optischen Detektion oder enzymatischer Amplifikation. DNA - bindende oder organisierende Proteine (z.B. endogene Transkriptionsfaktoren) bieten im Kontrast zu den Hybridisierungsreaktionen eine überaus interessante Alternative um dsDNA direkt und zugleich spezifisch zu detektieren oder diese aus einem komplexen Gemisch heraus anzureichern. Im Rahmen der Entwicklung von neuartigen NAD - Assays zur direkten Detektion oder Anreicherung von Nukleinsäuren wurden vier DNA - bindende Proteine kloniert und in HEK293 und E. coli exprimiert. Der Cys2His2 - Zinkfinger (ZFD) vom humanen Transkriptionsfaktor Sp1 wurde mit MBP und 9×Lys - MBP fusioniert. Das MBP - Derivat 9×Lys - MBP ist eine erweiterte Variante mit neun aufeinanderfolgenden Lysinen im N - terminalen Bereich, welche eine regioselektive Immobilisierung ermöglichen soll. Der humane Sp3 - ZFD wurde mit EGFP fusioniert. Die Mitglieder der Sp - Familie binden spezifisch die Konsensussequenz 5’ - GGG GCG GGG - 3’ (GC - Box). Zusätzlich wurde die C - terminale DNA - bindende Domäne der E. coli DNA - Gyrase Untereinheit A (gyrA - CTD) ebenfalls mit MBP fusioniert. Die Domäne bindet spezifisch repetitive extragene Palindrome (REP), welche bislang nur auf bakteriellen Chromosomen vorkommen. Sämtliche MBP - Fusionsproteine liegen nach der Expression löslich vor und konnten über eine native Strategie aufgereinigt werden. Transiente Transfektionsexperimente in HEK293 zeigten einen destabilisierenden Effekt der Sp3 - ZFD und eine massive einhergehende Degradierung des EGFP - Fusionsproteins nach 120 h. Die Analyse der mRNA - Integrität nach Transfektion des Expressionsplasmids, sowie zellbiologische und proteinbiochemische Untersuchungen mit Durchflusszytometrie bzw. Western Blots deuten auf eine posttranslationale Modulierung von EGFP - Sp3 hin. Um die Hypothese der proteasomalen Degradierung von EGFP - Sp3 zu belegen, wurden transfizierte HEK293 mit dem reversiblen Proteasominhibitor MG132 behandelt. In Gegenwart von 1 µM MG132 konnte das zytosolische Fusionsprotein stabilisiert werden. Die hier präsentierten Daten offenbaren die humane Sp3 - ZFD als ein neues Substrat für das 26S - Proteasom. Lediglich die SUMOylierung von Wildtyp - Sp3 im Bereich der inhibitorischen Domäne (ID) ist bislang beschrieben worden. Die Funktionalität, Affinität und kinetische Parameter der mit MBP fusionierten Sp1 - ZFD und gyrA - CTD wurden anhand von Oberflächenplasmonresonanz (BIAcore) bzw. EMSAs analysiert. Sämtliche gewonnenen MBP - Fusionsproteine sind funktionell und interagieren mit dsDNA. Fusionsproteine mit Sp1 - Domäne zeigten in EMSAs ebenso eine Bindung an unspezifische dsDNA. In sensitiveren BIAcore - Assays mit immobilisierter dsDNA wurden (um den Faktor 2) geringere Assoziations (ka) - und Dissoziationsraten (kd) von MBP - Sp1 ermittelt, wenn bestimmte Basen innerhalb der GC - Box ausgetauscht wurden. Die Affinität (Kd) von MBP - Sp1 mit 4×10 - 9 M zur GC - Box und deren Derivate ist vergleichbar mit der Kd von nativem Sp1. Die EMSA - Experimente für MBP - gyrA zeigen eine deutliche Präferenz zum spezifischen dsDNA - Oligo in Gegenwart von humaner gDNA, eine interessante Eigenschaft die durchaus zur Anwendung in einem Assay zur Anreicherung von bakterieller DNA dienen kann. Nach der vorausgehenden Charakterisierung der MBP - Fusionsproteine wurden diese auf verschiedenen gängigen festen und semifesten Substraten über physische Adsorption, kovalent oder Affinität immobilisiert um das Konzept der direkten Detektion von dsDNA mit funktionellen Proteinen als neuartige Komponente in NAD - Assays umzusetzen. Lediglich MBP - Sp1 zeigte auf Glas und Polystyren - Mikrotiterplatten nach kovalenter oder adsorptiver Immobilisierung eine ausgeprägte Funktionalität hinsichtlich der Bindung von dsDNA. Die Immobilisierung von 9×Lys - MBP - Sp1 über identische Strategien führten zum massiven Verlust der ZFD - Funktion. Aus dieser Datenlage heraus wurde erfolgreich ein simples Lumineszenz - basiertes Mikrotiterplatten - Assay mit MBP - Sp1 entwickelt um PCR - Amplikons direkt aus einer analytischen PCR auf gDNA von S. aureus, welche die GC - Box beinhalten, nachzuweisen. Das spezifische Amplikon konnte mittels des simplen Assays in Gegenwart von 100fachem Überschuss an humaner gDNA nachgewiesen werden. Mit einem höheren Anteil an humaner gDNA wurde die PCR massiv inhibiert, ein negativer Effekt der bislang im Bereich der diagnostischen NAD - Assays nicht optimal adressiert wurde. Die magnetische Separation von bakterieller und humaner gDNA wurde dazu mit MBP - gyrA umgesetzt. Zunächst erfolgte die regioselektive Immobilisierung von MBP - gyrA auf Protein A - funktionalisierte magnetische Nanopartikel mittels MBP - Antikörper, wodurch die Funktionalität hinsichtlich der Bindung von dsDNA gewährleistet werden konnte. Dieses System eignet sich insbesondere für die Separation von bakterieller DNA (E. coli oder S. aureus) aus einem komplexen Gemisch mit bis zu 100fachem Überschuss an humaner gDNA. Die Kombination von MBP - gyrA - basierter magnetischer Separation mit NAD - Assays könnte deren Sensitivität signifikant erhöhen. Durch simple Verfahrensweise bietet das System einen wesentlichen Beitrag zur Verringerung des zeitlichen Aufwands für die Generierung therapierelevanter Resultate. / Methods for direct detection or enrichment of double - stranded DNA (dsDNA) possess tremendous potential for use in molecular diagnostics. Already established methods for nucleic acid detection (NAD) are generally based on the hybridization of two complementary strands followed by optical detection or enzymatic amplification. In contrast, DNA - binding or organizing proteins (e.g. endogenous transcriptions factors) are able to read the sequence information directly from dsDNA without prior denaturation of the double strand and subsequent hybridization. In order to develop novel NAD assays or assays for sample preparation, four artificial DNA - binding proteins were cloned, expressed and purified in HEK293 cells or E. coli. The Cys2His2 zinc finger domains (ZFD) from human Sp1 were fused to maltose binding protein (MBP) and its derivate 9×Lys - MBP, an extended variant with nine successive lysine residues in the N - terminal region of the protein to facilitate site - directed immobilization. The human Sp3 - ZFD was fused to green fluorescent protein (EGFP). The family of Sp - transcription factors was known to bind specifically the consensus sequence 5\'' - GGG GCG GGG - 3 \''(GC - box). Moreover, the C - terminal DNA - binding domain of E. coli DNA Gyrase subunit A (gyrA - CTD) was fused to MBP. The CTD binds specifically repetitive extragenic palindromes (REP), which were only found on prokaryotic chromosomes. All MBP fusion proteins were soluble after expression and could be purified to homogeneity. Surprisingly, transient transfection experiments in HEK293 revealed a destabilizing effect of the Sp3 - ZFD accompanied by massive degradation of the EGFP fusion protein after 120 h post transfection. Analysis of mRNA integrity in combination with western blots indicates a posttranslational modulation of EGFP - Sp3. To confirm the hypothesis of proteasomal degradation of EGFP - Sp3, transfected cells were treated with the reversible proteasome inhibitor MG132. In the presence of 1µM MG132 the fusion protein could be stabilized. Taken together, the data presented here identified the human Sp3 - CTD as a new substrate for the 26S proteasome. Only SUMOylation of wild type human Sp3 within the inhibitory domain (ID) has been described so far. Initial EMSA experiments showed that purified MBP - ZFD fusion proteins were functional in terms of interacting with dsDNA containing the specific sequence motiv. However, all proteins bound to unspecific dsDNA as well. Therefore MBP - Sp1 was subjected to BIAcore analysis to determine the rate constants for association ka, dissociation kd and the dissociation constant Kd of the GC - Box - Protein complex as well as mutants of the GC - Box. The determined Kd (4 × 10 - 9 M) for MBP - Sp1 associated with GC - box or its derivatives were found to be comparable with the Kd of native Sp1, however the rate constants were reduced 2 fold in presence of the modified GC - boxes. EMSA experiments with MBP - gyrA revealed functionality and a clear preference for specific dsDNA in the presence of unspecific human genomic DNA (gDNA). After preliminary functional characterization, MBP fusion proteins were immobilized by physical adsorption, covalent or by affinity on various solid substrates or nanoscaled magnetic beads to implement the concept of direct detection of dsDNA or specific enrichment of bacterial DNA, respectively. MBP - Sp1 remains functional after adsorptive or covalent immobilization on different chemical modified glas surfaces. 9×Lys - MBP - Sp1 shows significantly reduced functionality after immobilization on the same glas substrates by similar strategies. Moreover, a simple NAD - assay with adsorptive immobilized MBP - Sp1 on polystyrene in microtiter format was established for direct detection of GC - boxes within PCR - products from S. aureus gDNA. By using the assay, specific PCR - products could be detected in presence up to 100 - fold excess of human gDNA in relation to 10 ng bacterial DNA. Separation of bacterial DNA from human DNA from clinical samples may have an important impact on downstream applications, involving NAD assays. To address this often underestimated technical problem, a new functional protein MBP - gyrA was introduced to overcome some limitations of already established methods. MBP - gyrA was site - directed coupled on nanoscaled magnetic beads by affinity. This system enabled the fast and specific separation of gDNA of E. coli or S.aureus from a huge background of human gDNA. The combination of MBP - gyrA - based magnetic separation with NAD assays could significantly increase the sensitivity and shorten the time for initiation of effective treatment.

Zinc is an essential cofactor of several proteins. The roles of zinc in these proteins are classified as catalytic, structural or regulatory. Zinc present in structural sites is considered to be a chemically inert, static structural element. On the contrary, previous studies on a C2H2 type zinc finger model compound and the C3H type HIV-1 NCp7 C-terminal zinc knuckle have shown that zinc at these sites can undergo coordination sphere expansion under the influence of a Pt based electrophile. The pentacoordination observed around zinc in these experiments raises an important question: are the structural zinc motifs found in the proteins susceptible to coordination sphere expansion? Through DFT modeling, the existence and nature of the five coordinate zinc species was investigated. mPW1PW91 was chosen as the DFT method by benchmarking against the experimental parameters of a molecule that closely resembles those to be modeled. The results suggest that the observed coordination sphere expansion is due to the flexible nature of thiolate and chloride ligands that are part of the structure. However, if certain conditions are not met, the same flexibility can lead to the destabilization of these rather fragile structures. Unlike the stable three or four coordinate catalytic and structural zinc sites, at regulatory sites, zinc is typically bound to one or two protein ligands. Zinc inhibition of caspases which are central to the process of apoptosis is one such scenario. Several of the caspases are inhibited by zinc at low micromolar range. Regulation of caspases is a strategy for drug development toward apoptosis related diseases; thus it is important to know the molecular details of zinc inhibition of caspases. Currently, it is speculated that zinc binds to the active site His and Cys residues of caspases thus competing with the substrate. However our studies on caspase-3, using enzyme kinetics and biophysical methods, imply more than one zinc binding sites. Contrary to current beliefs, more than 50% of the inhibition is achieved by zinc without affecting substrate binding. Using DFT models, an alternative inhibitory zinc binding site, which better fits our experimental observations, is predicted.