Epäpuhtauksien vaikutus monokaliumfosfaattikiteen kasvuun

Työssä on tutkittu epäpuhtauksien vaikutusta kastelulannoitesuolojen monokaliumfosfaatin, kaliumnitraatin ja ureafosfaatin kiteytyksessä. Kirjallisuusosassa on käsitelty kastelulannoiteprosessit ja epäpuhtauksien vaikutus kastelulannoitteiden valmistuksessa. Kiteytys ja kiteenkasvu on esitetty perusyhtälöin, joissa on otettu epäpuhtauksien vaikutus huomioon. Tarkemmin on perehdytty monokaliumfosfaatin kiteytykseen ja kolmenarvoisten kationeiden, Al3+, Fe3+ ja Cr3+, vaikutukseen kiteiden kasvuun. Kolmenarvoiset metalli-ionit adsorboituvat kiteen pintaan haitaten kiteenkasvua, mikä vaikuttaa erityisesti kiteen prismapinnan kasvuun. Lisäksi on esitelty muita kiteenkasvuun vaikuttavia olosuhteita. Lopuksi on käsitelty kompleksinmuodostajia metalli-ionien haitallisten vaikutusten ehkäisijöinä. Kokeellisessa osassa suoritettiin liukoisuuskokeita monokaliumfosfaatin liukoisuuden selvittämiseksi eri pH-olosuhteissa. Suoritetuissa yksikidekokeissa tutkittiin pH:n ja kolmenarvoisten kationeiden; Al3+, Fe3+ ja Cr3+, vaikutus monokaliumfosfaattikiteen pituus- ja leveyskasvuun ja kidemuotoon eri ylikylläisyyksillä. Lisäksi tutkittiin voidaanko lämpötilaa ja pH muuttamalla tai pyrofosfaattia lisäämällä poistaa raudan kasvua inhiboima vaikutus. Kiteytyslämpötilaa nostamalla voidaan poistaa raudan haitallinen vaikutus kiteen kasvuun.

Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Unraveling the functional divergence of membrane-bound pyrophosphatases

Inorganic pyrophosphatases (PPases) are enzymes that hydrolyze pyrophosphate (PPi)which is produced as a byproduct in many important growth related processes e.g. in the biosynthesis of DNA, proteins and lipids. PPases can be either soluble or membranebound. Membrane-bound PPases (mPPases) are ion transporters that couple the energy released during PPi hydrolysis to Na+ or H+ transport. When I started the project, only three Na+-transporting mPPases were known to exist. In this study, I aimed to confirm if Na+-transport is a common function of mPPases. Furthermore, the amino acid residues responsible for determining the transporter specificity were unknown. I constructed a phylogenetic tree for mPPases and selected the representative bacterial and archaeal mPPases to be investigated. I expressed different prokaryotic mPPases in Escherichia coli, isolated these as inverted membrane vesicles and characterized their functions. In the first project I identified four new Na+-PPases, two K+-dependent H+-PPases and one K+-independent mPPase. The residues determining the transporter specificity were identified by site-directed mutagenesis. I showed that the conserved glutamate residues are important for specificity, though are not the only residues that influence it. This research clarified the ion transport specificities throughout the mPPase phylogenetic tree, and revealed that Na+ transport is a widespread function of mPPases. In addition, it became clear that the transporter specificity can be predicted from the amino acid sequence in combination with a phylogenetic analysis. In the second project, I identified a novel class of mPPases, which is capable of transporting both Na+ and H+ ions and is mainly found in bacteria of the human gastrointestinal tract. The physiological role of these novel enzymes may be to help the bacteria survive in the demanding conditions of the host. In the third project, I characterized the Chlorobium limicola Na+-PPase and found that this and related mPPases are able to transport H+ ions at subphysiological Na+ concentrations. In addition, the H+-transport activity was shown to be a common function of all studied Na+-PPases at low Na+ concentrations. I observed that mutating gate-lysine to asparagine eliminated the H+ but not the Na+ ion transport function, indicating the important role of the residue in the transport of H+. In the fourth project, I characterized the unknown and evolutionary divergent mPPase clade of the phylogenetic tree. The enzymes belonging to this clade are able to transport H+ ions and, based on their sequence, were expected to be K+- and Na+-independent. The sequences of membrane-bound PPase are usually highly conserved, but the enzymes belonging to this clade are more divergent and usually contain 100−150 extra amino acid residues compared to other known mPPases. Despite the vast sequence differences, these mPPases have the full set of important residues and, surprisingly, are regulated by Na+ and K+ ions. These enzymes are mainly of bacterial origin.