Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone Derivatives As Potent Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB)

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of naphthylchalcones has been identified as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In this work, 100 chalcones were designed, synthesized, and investigated for their inhibitory properties against MtbPtps. Structure-activity relationships (SAR) were developed, leading to the discovery of new potent inhibitors with IC50 values in the low-micromolar range. Kinetic studies revealed competitive inhibition and high selectivity toward the Mtb enzymes. Molecular modeling investigations were carried out with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of inhibitors as potential anti-TB drugs.

The protein LJM 111 from Lutzomyia longipalpis Salivary Gland Extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-alpha and IFN-gamma released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry. TNF-alpha and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-alpha and IFN-gamma. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-alpha whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils. (C) 2012 Elsevier B.V. All rights reserved.

Identification of protein expression signatures in gastric carcinomas using clustering analysis

Background and Aim: The identification of gastric carcinomas (GC) has traditionally been based on histomorphology. Recently, DNA microarrays have successfully been used to identify tumors through clustering of the expression profiles. Random forest clustering is widely used for tissue microarrays and other immunohistochemical data, because it handles highly-skewed tumor marker expressions well, and weighs the contribution of each marker according to its relatedness with other tumor markers. In the present study, we e identified biologically- and clinically-meaningful groups of GC by hierarchical clustering analysis of immunohistochemical protein expression. Methods: We selected 28 proteins (p16, p27, p21, cyclin D1, cyclin A, cyclin B1, pRb, p53, c-met, c-erbB-2, vascular endothelial growth factor, transforming growth factor [TGF]-beta I, TGF-beta II, MutS homolog-2, bcl-2, bax, bak, bcl-x, adenomatous polyposis coli, clathrin, E-cadherin, beta-catenin, mucin (MUC) 1, MUC2, MUC5AC, MUC6, matrix metalloproteinase [ MMP]-2, and MMP-9) to be investigated by immunohistochemistry in 482 GC. The analyses of the data were done using a random forest-clustering method. Results: Proteins related to cell cycle, growth factor, cell motility, cell adhesion, apoptosis, and matrix remodeling were highly expressed in GC. We identified protein expressions associated with poor survival in diffuse-type GC. Conclusions: Based on the expression analysis of 28 proteins, we identified two groups of GC that could not be explained by any clinicopathological variables, and a subgroup of long-surviving diffuse-type GC patients with a distinct molecular profile. These results provide not only a new molecular basis for understanding the biological properties of GC, but also better prediction of survival than the classic pathological grouping.

Physical-chemical, thermal, and functional properties of achira (Canna indica L.) flour and starch from different geographical origin

Achira (Canna indica L.) is a plant native to the Andes in South America, a starchy source, and its cultivation has expanded to different tropical countries, like Brazil. In order to evaluate the potential of this species, starch and flours with different particle size were obtained from Brazilian achira rhizomes. Proximal analyses, size distribution, SEM, swelling power, solubility, DSC, XRD analysis, and FTIR were performed for characterization of these materials. Flours showed high dietary fiber content (16.532.2% db) and high concentration of starch in the case of the smaller particle size fraction. Significant differences in protein and starch content, swelling power, solubility, and thermal properties were observed between the Brazilian and the Colombian starch. All the studied materials displayed the B-type XRD pattern with relative crystallinity of 20.1% for the flour and between 27.0 and 28.0% for the starches. Results showed that the starch and flour produced from achira rhizomes have great technological potential for use as functional ingredient in the food industry.

Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein

BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrPC). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrPC. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrPC-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrPC signaling

The study the properties structurals and elastics of phases lamellar of lipid.

Lamellar systems composed of lipid bilayers have been widely used as model system for investigating properties of biological membranes, interactions between membranes and with biomolecules. The composition of the membrane determines its three dimensional shape and its properties such as rigidity and compressibility which play an important role on membrane fusion, protein adhesion, interactions between proteins, etc. We present a systematic study of a lamellar system composed of lecithin and a commercial co-surfactant (Simusol), which is a mixture of ethoxylated fatty acids. Using X ray scattering and a new procedure to fit X-ray experimental data, we determine relevant parameters characterizing the lamellar structure, varying membrane composition from 100% of lecithin to 100% of Simulsol. We present experimental data illustrating the swelling behavior for the membrane of different compositions and the respective behavior of the Caillé parameter. From and GISAXS experiments on oriented films under controlled humidity we investigate the compressibility of the lamellar phase and the effect of incorporating co-surfactant. Combining the Caillé parameter and compressibility studies we determine the bending rigidity of membranes. The results obtained with this experimental approach and new procedure to fit X-ray experimental allows us to identify structural changes in the bilayer depending both on hydration and co-surfactant content, with implications on elastic properties of membranes.

Theoretical studies of Membrane Proteins : Properties, Prediction Methods and Genome-wide analysis

Membrane proteins are a large and important class of proteins. They are responsible for several of the key functions in a living cell, e.g. transport of nutrients and ions, cell-cell signaling, and cell-cell adhesion. Despite their importance it has not been possible to study their structure and organization in much detail because of the difficulty to obtain 3D structures. In this thesis theoretical studies of membrane protein sequences and structures have been carried out by analyzing existing experimental data. The data comes from several sources including sequence databases, genome sequencing projects, and 3D structures. Prediction of the membrane spanning regions by hydrophobicity analysis is a key technique used in several of the studies. A novel method for this is also presented and compared to other methods. The primary questions addressed in the thesis are: What properties are common to all membrane proteins? What is the overall architecture of a membrane protein? What properties govern the integration into the membrane? How many membrane proteins are there and how are they distributed in different organisms? Several of the findings have now been backed up by experiments. An analysis of the large family of G-protein coupled receptors pinpoints differences in length and amino acid composition of loops between proteins with and without a signal peptide and also differences between extra- and intracellular loops. Known 3D structures of membrane proteins have been studied in terms of hydrophobicity, distribution of secondary structure and amino acid types, position specific residue variability, and differences between loops and membrane spanning regions. An analysis of several fully and partially sequenced genomes from eukaryotes, prokaryotes, and archaea has been carried out. Several differences in the membrane protein content between organisms were found, the most important being the total number of membrane proteins and the distribution of membrane proteins with a given number of transmembrane segments. Of the properties that were found to be similar in all organisms, the most obvious is the bias in the distribution of positive charges between the extra- and intracellular loops. Finally, an analysis of homologues to membrane proteins with known topology uncovered two related, multi-spanning proteins with opposite predicted orientations. The predicted topologies were verified experimentally, providing a first example of "divergent topology evolution".

Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.

Molecular interactions: metal ions and protein chaperones in the urease system from Helicobacter pylori

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

New sensing platforms based on nanoscopic devices for probing protein-membrane interactions

A novel nanosized and addressable sensing platform based on membrane coated plasmonic particles for detection of protein adsorption using dark field scattering spectroscopy of single particles has been established. To this end, a detailed analysis of the deposition of gold nanorods on differently functionalized substrates is performed in relation to various factors (such as the pH, ionic strength, concentration of colloidal suspension, incubation time) in order to find the optimal conditions for obtaining a homogenous distribution of particles at the desired surface number density. The possibility of successfully draping lipid bilayers over the gold particles immobilized on glass substrates depends on the careful adjustment of parameters such as membrane curvature and adhesion properties and is demonstrated with complementary techniques such as phase imaging AFM, fluorescence microscopy (including FRAP) and single particle spectroscopy. The functionality and sensitivity of the proposed sensing platform is unequivocally certified by the resonance shifts of the plasmonic particles that were individually interrogated with single particle spectroscopy upon the adsorption of streptavidin to biotinylated lipid membranes. This new detection approach that employs particles as nanoscopic reporters for biomolecular interactions insures a highly localized sensitivity that offers the possibility to screen lateral inhomogeneities of native membranes. As an alternative to the 2D array of gold nanorods, short range ordered arrays of nanoholes in optically transparent gold films or regular arrays of truncated tetrahedron shaped particles are built by means of colloidal nanolithography on transparent substrates. Technical issues mainly related to the optimization of the mask deposition conditions are successfully addressed such that extended areas of homogenously nanostructured gold surfaces are achieved. Adsorption of the proteins annexin A1 and prothrombin on multicomponent lipid membranes as well as the hydrolytic activity of the phospholipase PLA2 were investigated with classical techniques such as AFM, ellipsometry and fluorescence microscopy. At first, the issues of lateral phase separation in membranes of various lipid compositions and the dependency of the domains configuration (sizes and shapes) on the membrane content are addressed. It is shown that the tendency for phase segregation of gel and fluid phase lipid mixtures is accentuated in the presence of divalent calcium ions for membranes containing anionic lipids as compared to neutral bilayers. Annexin A1 adsorbs preferentially and irreversibly on preformed phosphatidylserine (PS) enriched lipid domains but, dependent on the PS content of the bilayer, the protein itself may induce clustering of the anionic lipids into areas with high binding affinity. Corroborated evidence from AFM and fluorescence experiments confirm the hypothesis of a specifically increased hydrolytic activity of PLA2 on the highly curved regions of membranes due to a facilitated access of lipase to the cleavage sites of the lipids. The influence of the nanoscale gold surface topography on the adhesion of lipid vesicles is unambiguously demonstrated and this reveals, at least in part, an answer for the controversial question existent in the literature about the behavior of lipid vesicles interacting with bare gold substrates. The possibility of formation monolayers of lipid vesicles on chemically untreated gold substrates decorated with gold nanorods opens new perspectives for biosensing applications that involve the radiative decay engineering of the plasmonic particles.

Topological aspects of the human protein interaction network

It is currently widely accepted that the understanding of complex cell functions depends on an integrated network theoretical approach and not on an isolated view of the different molecular agents. Aim of this thesis was the examination of topological properties that mirror known biological aspects by depicting the human protein network with methods from graph- and network theory. The presented network is a partial human interactome of 9222 proteins and 36324 interactions, consisting of single interactions reliably extracted from peer-reviewed scientific publications. In general, one can focus on intra- or intermodular characteristics, where a functional module is defined as "a discrete entity whose function is separable from those of other modules". It is found that the presented human network is also scale-free and hierarchically organised, as shown for yeast networks before. The interactome also exhibits proteins with high betweenness and low connectivity which are biologically analyzed and interpreted here as shuttling proteins between organelles (e.g. ER to Golgi, internal ER protein translocation, peroxisomal import, nuclear pores import/export) for the first time. As an optimisation for finding proteins that connect modules, a new method is developed here based on proteins located between highly clustered regions, rather than regarding highly connected regions. As a proof of principle, the Mediator complex is found in first place, the prime example for a connector complex. Focusing on intramodular aspects, the measurement of k-clique communities discriminates overlapping modules very well. Twenty of the largest identified modules are analysed in detail and annotated to known biological structures (e.g. proteasome, the NFκB-, TGF-β complex). Additionally, two large and highly interconnected modules for signal transducer and transcription factor proteins are revealed, separated by known shuttling proteins. These proteins yield also the highest number of redundant shortcuts (by calculating the skeleton), exhibit the highest numbers of interactions and might constitute highly interconnected but spatially separated rich-clubs either for signal transduction or for transcription factors. This design principle allows manifold regulatory events for signal transduction and enables a high diversity of transcription events in the nucleus by a limited set of proteins. Altogether, biological aspects are mirrored by pure topological features, leading to a new view and to new methods that assist the annotation of proteins to biological functions, structures and subcellular localisations. As the human protein network is one of the most complex networks at all, these results will be fruitful for other fields of network theory and will help understanding complex network functions in general.

Characterization of the Staphylococcus aureus bone sialoprotein-binding protein SdrE and the serine protease EpiP

In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.

Das wasserlösliche Chlorophyll-Protein (WSCP): biochemische Charakterisierung und Untersuchungen zur biologischen Funktion

Das WSCP (water-soluble chlorophyll protein) der Brassicaceen ist das einzig bekannte Chlorophyll-bindende Protein, welches keine Carotinoide bindet. Es ist ein wasserlösliches, ca. 80 kDa großes Homotetramer mit 1-4 gebundenen Chlorophyllen. Das Protein ist äußerst stabil und vermag die gebundenen Chlorophylle vor Photooxidation zu schützen. Seine Funktion in der Pflanze ist bis heute ein Rätsel und sollte in dieser Arbeit zusammen mit seinen biochemischen Eigenschaften weiter aufgeklärt werden. Es wurden Versuche durchgeführt mit nativem und rekombinantem WSCP aus Blumenkohl (BoWSCP bzw. BoWSCPhis) und aus Arabidopsis thaliana (AtWSCP bzw. AtWSCPhis). Die Expressionsausbeute von BoWSCPhis konnte verbessert werden und zusätzlich wurde die Rekonstitutionsmethode für das rekombinante WSCP optimiert, sodass das pigmentierte Protein mit hoher Ausbeute und großer Reinheit gewonnen werden konnte. Zudem wurde ein neuer WSCP-Klon hergestellt, mBoWSCPhis, der in seiner Sequenz dem maturen nativen BoWSCP entspricht und weitaus weniger Aggregationsprobleme zeigte als BoWSCPhis. Weiterführende Versuche zur Stabilität und dem Oligomerisierungsgrad von WSCP haben die neue Erkenntnis erbracht, dass die Phytolschwänze der von WSCP gebundenen Chlorophylle zwar essentiell sind für die Stabilität von WSCP-Oligomeren, nicht aber für die Oligomerisierung selbst, wie es in der Literatur bislang postuliert wurde. Zusätzlich zu ihrer außerordentlichen Hitzestabilität erwiesen sich die Chl-WSCP-Komplexe als stabil in einem breiten pH-Spektrum. AtWSCPhis besaß eine vergleichbare Stabilität, und auch das Oligomerisierungsverhalten zeigte Ähnlichkeiten zu BoWSCPhis. Im Rahmen einer Forschungskooperation mit dem Institut für Optik und Atomare Physik der TU Berlin wurden zeitaufgelöste Absorptionsspektren sowie Tieftemperatur-Fluoreszenzspektren an Chl-WSCP-Komplexen gemessen. Die Ergebnisse zeigten deutlich, dass die WSCP-gebundenen Chlorophylle excitonisch gekoppelt sind und wiesen zudem auf unterschiedliche Chl-Bindungsmodi hin. Aufgrund seines einfachen Aufbaus und seines geringen Chlorophyllgehalts hat sich WSCP bei diesen Versuchen als sehr geeignetes Modellsystem erwiesen, um Messungen zur Chlorophyllbindung mit Vorhersagen aus theoretischen Modellen zu vergleichen. Bei den Experimenten zur biologischen Funktion wurden einerseits Arabidopsis thaliana WSCP-„knock-out“-Pflanzen unter verschiedenen Bedingungen charakterisiert, andererseits wurden Experimente mit rekombinantem WSCP durchgeführt, um eine mögliche Interaktion mit anderen Proteinen zu detektieren. Die vegetativen Stadien der Mutante zeigten keinen Phänotyp; das native Arabidopsis-WSCP konnte später bei der Wildtyp-Pflanze ausschließlich in jungen Schoten lokalisiert werden, was eine Erklärung hierfür lieferte. Rekombinantes WSCP konnte Chlorophylle aus nativem LHCII entfernen, eine Interaktion mit Chlorophyllase konnte jedoch nicht nachgewiesen werden; daher konnte auch die Hypothese, WSCP sei ein Chl-Carrier beim Chl-Abbau, nicht untermauert werden. Bei den durchgeführten Enzym-Assays wurde eine geringfügige Inhibition der Cysteinprotease Papain beobachtet, aber keine Inhibition der Serinprotease Trypsin, obwohl Blumenkohl-WSCP N-proximal das Motiv der Künitz-Proteaseinhibitoren besitzt. Die Frage nach der biologischen Funktion von WSCP bleibt also weiterhin offen.

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rn