Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme action

The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerabl plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by cytosolic proteins - real or artifact

The inhibitory effect of FeSO4-dependent cytosolic protein on microsomal HMGCoA reductase is on the enzyme activity and not an artifact of loss of the product, mevalonate, through phosphorylation, unlike that of ATP.Mg effect.

Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus.

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2.

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (MF) in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT.

Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

Decoration of Pasteurella multocida lipopolysaccharide with phosphocholine is important for virulence

Phosphocholine (PCho) is an important substituent of surface structures expressed by a number of bacterial pathogens. Its role in virulence has been investigated in several species, in which it has been shown to play a role in bacterial adhesion to mucosal surfaces, in resistance to antimicrobial peptides, or in sensitivity to complement-mediated killing. The lipopolysaccharide (LPS) structure of Pasteurella multocida strain Pm70, whose genome sequence is known, has recently been determined and does not contain PCho. However, LPS structures from the closely related, virulent P. multocida strains VP161 and X-73 were shown to contain PCho on their terminal galactose sugar residues. To determine if PCho was involved in the virulence of P. multocida, we used subtractive hybridization of the VP161 genome against the Pm70 genome to identify a four-gene locus (designated pcgDABC) which we show is required for the addition of the PCho residues to LPS. The proteins predicted to be encoded by pcgABC showed identity to proteins involved in choline uptake, phosphorylation, and nucleotide sugar activation of PCho. We constructed a P. multocida VP161 pcgC mutant and demonstrated that this strain produces LPS that lacks PCho on the terminal galactose residues. This pcgC mutant displayed reduced in vivo growth in a chicken infection model and was more sensitive to the chicken antimicrobial peptide fowlicidin-1 than the wild-type P. multocida strain

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

The Interplay Between KSHV-encoded Viral Cyclin and CDK-inhibitors p27Kip1 and p21Cip1 -implications for Viral Lymphomagenesis

Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

To reactivate or not to reactivate : Control of KSHV lytic replication is essential for apoptosis in response to p53 restoration

Kaposi's sarcoma herpesvirus (KSHV) is an oncogenic human virus and the causative agent of three human malignancies: Kaposi's sarcoma (KS), Multicentric Castleman's Disease (MCD), and primary effusion lymphoma (PEL). In tumors, KSHV establishes latent infection during which it produces no infectious particles. Latently infected cells can enter the lytic replication cycle, and upon provision of appropriate cellular signals, produce progeny virus. PEL, commonly described in patients with AIDS, represents a diffuse large-cell non-Hodgkin's lymphoma, with median survival time less than six months after diagnosis. As tumor suppressor gene TP53 mutations occur rarely in PEL, the aim of this thesis was to investigate whether non-genotoxic activation of the p53 pathway can eradicate malignant PEL cells. This thesis demonstrates that Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction, efficiently restored p53 function in PEL cells, leading to cell cycle arrest and massive apoptosis. Furthermore, we found that KSHV infection activated DNA damage signaling, rendering the cells more sensitive to p53-dependent cell death. We also showed in vivo the therapeutic potential of p53 restoration that led to regression of subcutaneous and intraperitoneal PEL tumor xenografts without adversely affecting normal cells. Importantly, we demonstrated that in a small subset of intraperitoneal PEL tumors, spontaneous induction of viral reactivation dramatically impaired Nutlin-3-induced p53-mediated apoptosis. Accordingly, we found that elevated KSHV lytic transcripts correlated with PEL tumor burden in animals and that inhibition of viral reactivation in vitro restored cytotoxic activity of a small-molecule inhibitor of the p53-MDM2 interaction. Latency provides a unique opportunity for KSHV to escape host immune surveillance and to establish persistent infections. However, to maintain viral reservoirs and spread to other hosts, KSHV must be reactivated from latency and enter into the lytic growth phase. We showed that phosphorylation of nucleolar phosphoprotein nucleophosmin (NPM) by viral cyclin-CDK6 is critical for establishment and maintenance of the KSHV latency. In short, this study provides evidence that the switch between latent phase and lytic replication is a critical step that determines the outcome of viral infection and the pathogenesis of KSHV-induced malignancies. Our data may thus contribute to development of novel targeted therapies for intervention and treatment of KSHV-associated cancers.

p53 and DNA damage checkpoint responses in the human prostate

Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer mortality in men. In 2004, 5237 new cases were diagnosed and altogether 25 664 men suffered from prostate cancer in Finland (Suomen Syöpärekisteri). Although extensively investigated, we still have a very rudimentary understanding of the molecular mechanisms leading to the frequent transformation of the prostate epithelium. Prostate cancer is characterized by several unique features including the multifocal origin of tumors and extreme resistance to chemotherapy, and new treatment options are therefore urgently needed. The integrity of genomic DNA is constantly challenged by genotoxic insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, thus facilitating damage repair, apoptosis or cellular senescence. Cellular DNA damage triggers the activation of tumor suppressor protein p53 and Wee1 kinase which act as executors of the cellular checkpoint responses. These are essential for genomic integrity, and are activated in early stages of tumorigenesis in order to function as barriers against tumor formation. Our work establishes that the primary human prostatic epithelial cells and prostatic epithelium have unexpectedly indulgent checkpoint surveillance. This is evidenced by the absence of inhibitory Tyr15 phosphorylation on Cdk2, lack of p53 response, radioresistant DNA synthesis, lack of G1/S and G2/M phase arrest, and presence of persistent gammaH2AX damage foci. We ascribe the absence of inhibitory Tyr15 phosphorylation to low levels of Wee1A, a tyrosine kinase and negative regulator of cell cycle progression. Ectopic Wee1A kinase restored Cdk2-Tyr15 phosphorylation and efficiently rescued the ionizing radiation-induced checkpoints in the human prostatic epithelial cells. As variability in the DNA damage responses has been shown to underlie susceptibility to cancer, our results imply that a suboptimal checkpoint arrest may greatly increase the accumulation of genetic lesions in the prostate epithelia. We also show that small molecules can restore p53 function in prostatic epithelial cells and may serve as a paradigm for the development of future therapeutic agents for the treatment of prostate cancer We hypothesize that the prostate has evolved to activate the damage surveillance pathways and molecules involved in these pathways only to certain stresses in extreme circumstances. In doing so, this organ inadvertently made itself vulnerable to genotoxic stress, which may have implications in malignant transformation. Recognition of the limited activity of p53 and Wee1 in the prostate could drive mechanism-based discovery of preventative and therapeutic agents.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Molecular basis of the kidney filtration barrier : Role of the nephrin protein complex

The kidney filtration barrier consists of fenestrated endothelial cell layer, glomerular basement membrane and slit diaphragm (SD), the specialized junction between glomerular viscelar epithelial cells (podocytes). Podocyte injury is associated with the development of proteinuria, and if not reversed the injury will lead to permanent deterioration of the glomerular filter. The early events are characterized by disruption of the integrity of the SD, but the molecular pathways involved are not fully understood. Congenital nephrotic syndrome of the Finnish type (CNF) is caused by mutations in NPHS1, the gene encoding the SD protein nephrin. Lack of nephrin results in loss of the SD and massive proteinuria beginning before birth. Furthermore, nephrin expression is decreased in acquired human kidney diseases including diabetic nephropathy. This highlights the importance of nephrin and consequently SD in regulating the kidney filtration function. However, the precise molecular mechanism of how nephrin is involved in the formation of the SD is unknown. This thesis work aimed at clarifying the role of nephrin and its interaction partners in the formation of the SD. The purpose was to identify novel proteins that associate with nephrin in order to define the essential molecular complex required for the establishment of the SD. The aim was also to decipher the role of novel nephrin interacting proteins in podocytes. Nephrin binds to nephrin-like proteins Neph1 and Neph2, and to adherens junction protein P-cadherin. These interactions have been suggested to play a role in the formation of the SD. In this thesis work, we identified densin as a novel interaction partner for nephrin. Densin was localized to the SD and it was shown to bind to adherens junction protein beta-catenin. Furthermore, densin was shown to behave in a similar fashion as adherens junction proteins in cell-cell contacts. These results indicate that densin may play a role in cell adhesion and, therefore, may contribute to the formation of the SD together with nephrin and adherens junction proteins. Nephrin was also shown to bind to Neph3, which has been previously localized to the SD. Neph3 and Neph1 were shown to induce cell adhesion alone, whereas nephrin needed to trans-interact with Neph1 or Neph3 from the opposite cell surface in order to make cell-cell contacts. This was associated with the decreased tyrosine phosphorylation of nephrin. These data extend the current knowledge of the molecular composition of the nephrin protein complex at the SD and also provide novel insights of how the SD may be formed. This thesis work also showed that densin was up-regulated in the podocytes of CNF patients. Neph3 was up-regulated in nephrin deficient mouse kidneys, which share similar podocyte alterations and lack of the SD as observed in CNF patients podocytes. These data suggest that densin and Neph3 may have a role in the formation of morphological alterations in podocytes detected in CNF patients. Furthermore, this thesis work showed that deletion of beta-catenin specifically from adult mouse podocytes protected the mice from the development of adriamycin-induced podocyte injury and proteinuria compared to wild-type mice. These results show that beta-catenin play a role in the adriamycin induced podocyte injury. Podocyte injury is a hallmark in many kidney diseases and the changes observed in the podocytes of CNF patient share characteristics with injured podocytes observed in chronic kidney diseases. Therefore, the results obtained in this thesis work suggest that densin, Neph3 and beta-catenin participate in the molecular pathways which result in morphological alterations commonly detected in injured podocytes in kidney diseases.

Effect of lutein on the transport of Ca2+ across phospholipid bilayer and mitochondrial membrane

Lutein (3,3'-dihydroxy alpha-carotene), a xanthophyll present in plant chloroplasts, increases the permeability of phospholipid vesicles to Ca2+, even though the pigment does not bind the metal ion. Energy-dependent uptake of Ca2+ by mitochondria is inhibited by lutein, which permits a rapid efflux of the ion from Ca2+-loaded mitochondria. These results are consistent with the view that the deleterious action of lutein on mitochondrial oxidative phosphorylation results from its destabilizing action on membrane structure.

Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Activation of macrophages by interferon gamma (IFN- ) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN- -induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN- -treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN- -induced NO production, and they highlight the critical role of nirC as a virulence gene.