Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Influence of the Paracoccidioides brasiliensis14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensisremains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensismolecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensisuses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

GENETIC AND BIOCHEMICAL ANALYSIS OF THE TESTIS-SPECIFIC ZINC FINGER PROTEIN CTCFL/BORIS

Abstract en FrançaisCTCFL a d'abord été identifié comme un paralogue de la protéine ubiquitaire CTCF en raison de sa forte homologie entre leurs onze « zinc fingers », un domaine de liaison à l'ADN. Parmi ses nombreux rôles, la liaison des zinc fingers de CTCF à la région de contrôle de l'empreinte (ICR) maternelle non-méthylée Igf2/H19, contrôle l'expression empreinte (monoallélique) de H19 et IGF2 dans les cellules somatiques. La méthylation de l'ICR Igf2/H19 paternelle est nécessaire à l'expression empreinte de ces deux gènes. Bien que le mécanisme par lequel l'ICR est méthylé soit mal compris, il est connu que l'établissement de la méthylation se produit pendant le développement des cellules germinales mâles et que les ADN méthyltransférases de novo DNMT3A et DNMT3L sont essentiels. Par conséquent, CTCFL fournit un bon candidat pour un rôle dans la méthylation de l'ICR paternelle Igf2/H19 en raison de son expression restreinte à certains types de cellules où la méthylation de l'ICR a lieu (spermatogonies et spermatocytes) ainsi qu'en raison sa capacité à lier les ICR lgf2/HÎ9 dans ces cellules. Les premiers travaux expérimentaux de cette thèse portent sur le rôle possible des mutations de CTCFL chez les patients atteints du syndrome de Silver-Russell (SRS), où une diminution de la méthylation de l'ICR IGF2/H19 a été observée chez 60% d'entre eux. Admettant que CTCFL pourrait être muté chez ces patients, j'ai examiné les mutations possibles de CTCFL chez 35 d'entre eux par séquençage de l'ADN et analyse du nombre de copies d'exons. N'ayant trouvé aucune mutation chez ces patients, cela suggère que les mutations de CTCFL ne sont pas associées au SRS. Les travaux expérimentaux suivants ont porté sur les modifications post-traductionnelles de CTCFL par la protéine SU MO « small ubiquitin-like modifier » (SUMO). La modification de protéines par SU MO change les interactions avec d'autres molécules (ADN ou protéines). Comme CTCFL régule sans doute l'expression d'un certain nombre de gènes dans le cancer et que plusieurs facteurs de transcription sont régulés par SUMO, j'ai mené des expériences pour déterminer si CTCFL est sumoylé. En effet, j'ai observé que CTCFL est sumoylated in vitro et in vivo et j'ai déterminé les deux résidus d'attachement de SUMO aux lysines 181 et 645. Utilisant les mutants de CTCFL K181R et K645R ne pouvant pas être sumoylated, j'ai évalué les conséquences fonctionnelles de la modification par SUMO. Je n'ai trouvé aucun changement significatif dans la localisation subcellulaire, la demi-vie ou la liaison à l'ADN, mais ai constaté que la sumoylation module à la fois {'activation CTCFL-dépendante et la répression de l'expression génique. Il s'agit de la première modification post-traductionnelle décrite pour CTCFL et les conséquences possibles de cette modification sont discutées pour le cancer et les testicules normaux. Avec cette thèse, j'espère avoir ajouté des résultats importants à l'étude de CTCFL et donné quelques idées pour de futures recherches.AbstractJeremiah Bernier-Latmani, Institute of Pathology, University of Lausanne, CHUVCTCFL was first identified as a paralog of the ubiquitous protein CTCF because of high homology between their respective eleven zinc fingers, a DNA binding domain. Among its many roles, CTCF zinc finger-mediated binding to the unmethylated maternal Igf2/H19 imprinting control region (ICR), controls the imprinted (monoallelic) expression of Igf2 and H19 in somatic cells. Methylation of the paternal Igf2/H19 ICR is necessary for the imprinted expression of the two genes. Although the mechanism by which the ICR is methylated is incompletely understood, it is known that establishment of methylation occurs during male germ cell development and the de novo DNA methyltransferases DNMT3A and DNMT3L are essential. Therefore, CTCFL provided a good candidate to play a role in methylation of the paternal Igf2/H19 ICR because of its restricted expression to cell types where ICR methylation takes place (spermatogonia and spermatocytes) and its ability to bind the Igf2/H19 ICR in these cells. The first experimental work of this thesis investigated the possible role of CTCFL mutations in Silver-Russell syndrome (SRS) patients, where it has been observed that 60% of the patients have reduced methylation of the IGF2/HÎ9 ICR. Reasoning that CTCFL could be mutated in these patients, I screened 35 patients for mutations in CTCFL by DNA sequencing and exon copy number analysis, I did not find any mutations in these patients suggesting that mutations of CTCFL are not associated with SRS. The next experimental work of my thesis focused on posttranslational modification of CTCFL by small ubiquitin-like modifier (SUMO) protein. SUMO modification of proteins changes the interactions with other molecules (DNA or protein). As CTCFL arguably regulates the expression of a number of genes in cancer and many transcription factors are regulated by SUMO, I conducted experiments to assess whether CTCFL is sumoylated. I found that CTCFL is sumoylated in vitro and in vivo and determined the two residues of SUMO attachment to be lysines 181 and 645. Using K181R, K645R mutated CTCFL- which cannot be detected to be sumoylated-1 assessed the functional consequences of SUMO modification. I found no significant changes in subcellular localization, half-life or DNA binding, but found that sumoylation modulates both CTCFL-dependent activation and repression of gene expression. This is the first posttranslational modification described for CTCFL and possible consequences of this modification are discussed in both cancer and normal testis. With this thesis, I hope I have added important findings to the study of CTCFL and provide some ideas for future research.

Targeting the PI3K p110alpha isoform inhibits medulloblastoma proliferation, chemoresistance, and migration.

PURPOSE: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in medulloblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in medulloblastoma. EXPERIMENTAL DESIGN: The expression pattern and functions of class I(A) PI3K isoforms were investigated in medulloblastoma tumour samples and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110alpha, p110beta, or p110delta by means of RNA interference or inhibition with isoform-specific PI3K inhibitors. RESULTS: Overexpression of the catalytic p110alpha isoform was detected in a panel of primary medulloblastoma samples and cell lines compared with normal brain tissue. Down-regulation of p110alpha expression by RNA interference impaired the growth of medulloblastoma cells, induced apoptosis, and led to decreased migratory capacity of the cells. This effect was selective, because RNA interference targeting of p110beta or p110delta did not result in a comparable impairment of DAOY cell survival. Isoform-specific p110alpha inhibitors also impaired medulloblastoma cell proliferation and sensitized the cells to chemotherapy. Medulloblastoma cells treated with p110alpha inhibitors further displayed reduced activation of Akt and the ribosomal protein S6 kinase in response to stimulation with hepatocyte growth factor and insulin-like growth factor-I. CONCLUSIONS: Together, our data reveal a novel function of p110alpha in medulloblastoma growth and survival.

ß-Catenin Regulation during the Cell Cycle: Implications in G2/M and Apoptosis

ß-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. ß-Catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems ß-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of ß-Catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous ß-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase¿synchronized epithelial cells. ß-Catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of ß-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not ß-catenin, is detected in M phase. Interestingly, overexpression of a stable form of ß-catenin, or inhibition of endogenous ß-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for ß-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.

Toll-interacting protein modulates colitis susceptibility in mice.

BACKGROUND: The intestinal epithelium accommodates with a myriad of commensals to maintain immunological homeostasis, but the underlying mechanisms regulating epithelial responsiveness to flora-derived signals remain poorly understood. Herein, we sought to determine the role of the Toll/interleukin (IL)-1 receptor regulator Toll-interacting protein (Tollip) in intestinal homeostasis. METHODS: Colitis susceptibility was determined after oral dextran sulfate sodium (DSS) administration or by breeding Tollip on an IL-10 background. The intestinal flora was depleted with 4 antibiotics before DSS exposure to assess its contribution in colitis onset. Bone marrow chimeras were generated to identify the cellular compartment, whereby Tollip may negatively regulate intestinal inflammation in response to DSS. Tollip-dependent epithelial barrier functions were studied in vitro by using Tollip-knockdown in Caco-2 cells and in vivo by immunohistochemistry and fluorescein isothiocyanate-labeled dextran gavage. RESULTS: Genetic ablation of Tollip did not lead to spontaneous intestinal inflammatory disorders. However, Tollip deficiency aggravated spontaneous disease onset in IL-10 mice and increased susceptibility to DSS colitis. Increased colitis severity in Tollip-deficient mice was not improved by bacterial flora depletion using broad-spectrum antibiotics. In addition, DSS exposure of bone marrow chimeric mice revealed a protective role for Tollip in nonhematopoietic cells. Knockdown of Tollip in epithelial cells led to exaggerated NFκ-B activity and proinflammatory cytokine secretion. Finally, DSS-treated Tollip mice showed enhanced intestinal permeability and increased epithelial apoptosis when compared with wild-type controls, a finding that coincided with tight junction alterations on injury. CONCLUSION: Overall, our data show an essential role for Tollip on colitis susceptibility in mice.

HDLs protect the MIN6 insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress and without restoring ER functionality.

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells.

Rat pancreatic alpha- and beta-cells are critically dependent on hormonal signals generating cyclic AMP (cAMP) as a synergistic messenger for nutrient-induced hormone release. Several peptides of the glucagon-secretin family have been proposed as physiological ligands for cAMP production in beta-cells, but their relative importance for islet function is still unknown. The present study shows expression at the RNA level in beta-cells of receptors for glucagon, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide I(7-36) amide (GLP-I), while RNA from islet alpha-cells hybridized only with GIP receptor cDNA. Western blots confirmed that GLP-I receptors were expressed in beta-cells and not in alpha-cells. Receptor activity, measured as cellular cAMP production after exposing islet beta-cells for 15 min to a range of peptide concentrations, was already detected using 10 pmol/l GLP-I and 50 pmol/l GIP but required 1 nmol/l glucagon. EC50 values of GLP-I- and GIP-induced cAMP formation were comparable (0.2 nmol/l) and 45-fold lower than the EC50 of glucagon (9 nmol/l). Maximal stimulation of cAMP production was comparable for the three peptides. In purified alpha-cells, 1 nmol/l GLP-I failed to increase cAMP levels, while 10 pmol/l to 10 nmol/l GIP exerted similar stimulatory effects as in beta-cells. In conclusion, these data show that stimulation of glucagon, GLP-I, and GIP receptors in rat beta-cells causes cAMP production required for insulin release, while adenylate cyclase in alpha-cells is positively regulated by GIP.

Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.

Potentiation of glucose-induced insulin secretion by intestinal factors has been described for many years. Today, two major peptides with potent insulinotropic action have been recognized: gastric inhibitory peptide and truncated forms of glucagon-like peptide I, GLP-I(7-37) or the related GLP-I(7-36)amide. These hormones have specific beta-cell receptors that are coupled to production of cAMP and activation of cAMP-dependent protein kinase. Elevation in intracellular cAMP levels is required to mediate the glucoincretin effect of these hormones: the potentiation of insulin secretion in the presence of stimulatory concentrations of glucose. In addition, circulating glucoincretins maintain basal levels of cAMP, which are necessary to keep beta-cells in a glucose-competent state. Interactions between glucoincretin signaling and glucose-induced insulin secretion may result from the phosphorylation of key elements of the glucose signaling pathway by cAMP-dependent protein kinase. These include the ATP-dependent K+ channel, the Ca++ channel, or elements of the secretory machinery itself. In NIDDM, the glucoincretin effect is reduced. However, basal or stimulated gastric inhibitory peptide and glucagon-like peptide I levels are normal or even elevated, suggesting that signals induced by these hormones on the beta-cells are probably altered. At pharmacological doses, infusion of glucagon-like peptide I but not gastric inhibitory peptide, can ameliorate postprandial insulin secretory response in NIDDM patients. Agonists of the glucagon-like peptide I receptor have been proposed as new therapeutic agents in NIDDM.

Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway.

OBJECTIVE: The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) generates pancreatic beta-cells apoptosis mainly through activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced beta-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS: Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1 beta. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Ex-4 inhibited induction of the JNK pathway elicited by IL-1 beta. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1 beta. CONCLUSIONS: The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1 beta and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis.

Spermatogenesis relies on the precise regulation of the self-renewal and differentiation of spermatogonia to provide a continuous supply of differentiating germ cells. The understanding of the cellular pathways regulating this equilibrium remains unfortunately incomplete. This investigation aimed to elucidate the testicular and ovarian functions of the glucocorticoid-induced leucine zipper protein (GILZ) encoded by the X-linked Tsc22d3 (Gilz) gene. We found that GILZ is specifically expressed in the cytoplasm of proliferating spermatogonia and preleptotene spermatocytes. While Gilz mutant female mice were fully fertile, constitutive or male germ cell-specific ablation of Gilz led to sterility due to a complete absence of post-meiotic germ cells and mature spermatozoa. Alterations were observed as early as postnatal day 5 during the first spermatogenic wave and included extensive apoptosis at the spermatogonial level and meiotic arrest in the mid-late zygotene stage. Overall, these data emphasize the essential role played by GILZ in mediating spermatogonial survival and spermatogenesis.

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

Neuropeptide Y at the cellular level – Studies on PreproNPY L7P Polymorphism and the Mitochondrial Form of NPY

Neuropeptide Y (NPY) is a widely expressed neurotransmitter in the central and peripheral nervous systems. Thymidine 1128 to cytocine substitution in the signal sequence of the preproNPY results in a single amino acid change where leucine is changed to proline. This L7P change leads to a conformational change of the signal sequence which can have an effect on the intracellular processing of NPY. The L7P polymorphism was originally associated with higher total and LDL cholesterol levels in obese subjects. It has also been associated with several other physiological and pathophysiological responses such as atherosclerosis and T2 diabetes. However, the changes on the cellular level due to the preproNPY signal sequence L7P polymorphism were not known. The aims of the current thesis were to study the effects of the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes in primary cultured and genotyped human umbilical vein endothelial cells (HUVEC), in neuroblastoma (SK-N-BE(2)) cells and in fibroblast (CHO-K1) cells. Also, the putative effects of the L7P polymorphism on proliferation, apoptosis and LDL and nitric oxide metabolism were investigated. In the course of the studies a fragment of NPY targeted to mitochondria was found. With the putative mitochondrial NPY fragment the aim was to study the translational preferences and the mobility of the protein. The intracellular distribution of NPY between the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes was found to be different. NPY immunoreactivity was prominent in the [p.L7]+[p.P7] cells while the proNPY immunoreactivity was prominent in the [p.L7]+[p.L7] genotype cells. In the proliferation experiments there was a difference in the [p.L7]+[p.L7] genotype cells between early and late passage (aged) cells; the proliferation was raised in the aged cells. NPY increased the growth of the cells with the [p.L7]+[p.P7] genotype. Apoptosis did not seem to differ between the genotypes, but in the aged cells with the [p.L7]+[p.L7] genotype, LDL uptake was found to be elevated. Furthermore, the genotype seemed to have a strong effect on the nitric oxide metabolism. The results indicated that the mobility of NPY protein inside the cells was increased within the P7 containing constructs. The existence of the mitochondria targeted NPY fragment was verified, and translational preferences were proved to be due to the origin of the cells. Cell of neuronal origin preferred the translation of mature NPY (NPY1-36) when compared to the non neuronal cells that translated both, NPY and the mitochondrial fragment of NPY. The mobility of the mitochondrial fragment was found to be minimal. The functionality of the mitochondrial NPY fragment remains to be investigated. L7P polymorphism in the preproNPY causes a series of intracellular changes. These changes may contribute to the state of cellular senescence, vascular tone and lead to endothelial dysfunction and even to increased susceptibility to diseases, like atherosclerosis and T2 diabetes.

JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.