Umeå University's logo

Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni(2+) affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 11 of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.

The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.

The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.

The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.

During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.

The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution

synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.

Receptor tyrosine kinase (RTK) c-Kit signalling is crucial for the proliferation, survival and differentiation of haematopoietic stem cells (HSCs). To further understand the mechanisms underlying these events we explored how the downstream mediators interact. The present study investigated the function of conventional protein kinase Cs (c-PKC) in c-Kit mediated signalling pathways in HSC-like cell lines. This analysis supported earlier findings, that steel factor (SF) activates c-PKC, extracellular signal-regulated kinase (Erk) and protein kinase B (PKB). The present results were consistent with an important role of c-PKC in the positive activation of Erk and for proliferation. Further, it was observed that c-PKC negatively regulated PKB activity upon SF stimulation, indicating that c-PKC acts as a suppressor of c-Kit signalling. Finally, these observations were extended to show that c-PKC mediated the phosphorylation of the endogenous c-Kit receptor on serine 746, resulting in decreased overall tyrosine phosphorylation of c-Kit upon SF stimulation. This report showed that this specific feedback mechanism of c-PKC mediated phosphorylation of the c-Kit receptor has consequences for both proliferation and survival of HSC-like cell lines.

Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH).

To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe.

The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum.

The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.

Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.

Inhibitory receptors expressed on natural killer (NK) cells abrogate positive signals upon binding corresponding major histocompatibility complex (MHC) class I molecules on various target cells. By directly micromanipulating the effector-target cell encounter using an optical tweezers system which allowed temporal and spatial control, we demonstrate that Ly49-MHC class I interactions prevent characteristic cellular responses in NK cells upon binding to target cells. Furthermore, using this system, we directly demonstrate that an NK cell already bound to a resistant target cell may simultaneously bind and kill a susceptible target cell. Thus, although Ly49-mediated inhibitory signals can prevent many types of effector responses, they do not globally inhibit cellular function, but rather the inhibitory signal is spatially restricted towards resistant targets.

Queuosine (Q), a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora. However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C.elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation.

Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation.

We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils.

The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.

Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional "peak" in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome.

Painting of fourth (POF) is a chromosome-specific protein in Drosophila and represents the first example of an autosome-specific protein. POF binds to chromosome 4 in Drosophila melanogaster, initiating at the proximal region, followed by a spreading dependent on chromosome 4-specific sequences or structures. Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila, a complex including the male-specific lethal proteins, "paints" the male X chromosome, mediating its hypertranscription, explained to some extent by the acetylation of lysine 16 on histone H4. Here, we show that Pof is essential for viability in both sexes and for female fertility. POF binding to an autosome, the F element, is conserved in genus Drosophila, indicating functional conservation of the autosome specificity. In three of nine studied species, POF binds to the male X chromosome. When bound to the male X, it also colocalizes with the dosage compensation protein male-specific lethal 3, suggesting a relationship to dosage compensation. The chromosome specificity is determined at the species level and not by the amino acid sequence. We argue that POF is involved in a chromosome-specific regulatory function.

The Drosophila melanogaster gene Anaplastic Lymphoma Kinase (DAlk) is homologous to mammalian Alk, which is a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans the t(2;5) translocation involving the Alk locus encodes an active form of Alk that is the causative agent in Non-Hodgkin’s Lymphoma (Morris et al., 1994). Alk has also been associated with other cancers such as inflammatory myofibroblastic tumours (IMTs). The physiological function of the Alk RTK has not been described in any system until very recently, and is still not defined in vertebrates. The molecular similarity between Drosophila Alk and mammalian Alk suggested that mutation of Alk in flies may affect similar functional and developmental processes, and thus lead to some understanding of Alk function in vivo.

By employing an EMS mutagenesis screen we were able to obtain loss-of-function mutants in the Drosophila DAlk gene. Eleven independent DAlk mutants were identified and characterized. DAlk is normally expressed in the developing gut and in the CNS. DAlk mutant animals have a lethal phenotype and die at late embryonic stages or as 1st instar larva. In DAlk mutant embryos there is a complete failure in the development of the midgut whereas the CNS appears normal. The midgut consists of visceral musculature that is syncytial and is formed by fusion of multiple myoblasts. This is a dynamic process where two types of myoblasts, i.e. fusion-competent-myoblasts and founder-cells that function as seeds for muscle formation, fuse. In DAlk homozygous embryos there is no founder cell specification, which explains the failure of midgut formation in these embryos.

Recently a novel secreted molecule Jelly Belly (Jeb) was identified. Jeb is expressed in the tissue neighbouring the DAlk expressing cells of the developing visceral mesoderm. Jeb mutant embryos show a phenotype that is similar to that of DAlk mutant embryos. We have been able to show that Jeb is the ligand for DAlk in the developing visceral mesoderm and that Jeb binding stimulates a DAlk driven ERK signaling pathway. This leads to the expression of Dumbfounded (duf)/kin of Irregular chiasm-C (kirre), a founder-cell specific immunoglobulin that has an important role in myoblast aggregation and fusion.

The functional Drosophila midgut is made up of the visceral muscle that encircles the endodermal tube. This tube formation includes migration of cells originating in the anterior and posterior parts of the embryo, first along the anterior-posterior axis using the visceral mesoderm as a template, then dorsally and ventrally. In DAlk mutant embryos there is no visceral muscle fusion and both the visceral mesoderm and the endoderm fail to undergo dorsal-ventral migration.

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, which encodes a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans, the t(2;5) translocation, which involves the ALK locus, produces an active form of ALK, which is the causative agent in non-Hodgkin's lymphoma. The physiological function of the Alk RTK, however, is unknown. In this paper, we describe loss-of-function mutants in the Drosophila Alk gene that cause a complete failure of the development of the gut. We propose that the main function of Drosophila Alk during early embryogenesis is in visceral mesoderm development.

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

Much of the material taken into cells by endocytosis is rapidly returned to the plasma membrane by the endocytic recycling pathway. Although recycling is vital for the correct localization of cell membrane receptors and lipids, the molecular mechanisms that regulate recycling are only partially understood. Here we show that in C. elegans, endocytic recycling is inhibited by NUM-1A, the nematode Numb homologue. NUM-1A::GFP fusion protein is localized to the baso-lateral surfaces of many polarized epithelial cells including the hypodermis and the intestine. We show that increased NUM-1A levels cause morphological defects in these cells similar to those caused by loss-of-function mutations in rme-1, a positive regulator of recycling both in C. elegans and mammals. We describe the isolation of worms lacking num-1A activity and show that, consistent with a model in which NUM-1A negatively regulates recycling in the intestine, loss of num-1A function bypasses the requirement for RME-1. Genetic epistasis analysis with rab-10, which is required at an early part of the recycling pathway, suggests that loss of num-1A function does not affect the uptake of material by endocytosis but rather inhibits baso-lateral recycling downstream of rab-10.