Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, proinflammatory cytokines, trigger activation of MAPK pathway through phosphorylation on a TGY motif within the kinase activation loop. Protein MAPK appears to play a major role in apoptosis. It has been causally implicated in sepsis and arthritis. The translational small ubiquitin related modifier (SUMO) modification of proteins has been shown to play multiple functional roles in several cellular processes, including signal transduction, protein targeting, stabilization, transcriptional activation and apoptosis. Our previous study demonstrated that the expression levels of SUMO-1 rnRNA and proteins were enhanced in Helicobacter pylori infected human gastric epithelial cells. The activation of MAPK pathway and cellular apoptosis of AGS cell lines were increased during Helicobacter pylori infection. It was hypothesized that Helicobacter pylori functioning as a biological stress that induced MAPK mediated apoptosis which may be regulated by sumoylation. Results showed that MAPK phosphorylation and cellular apoptosis were enhanced in RFP-SUMOs or GFP-MAPK expressing cells, especially during Helicobacter pylori infection. It was inhibited by pretreatment of MAPK inhibitor. The enhanced phosphorylation and apoptosis were observed during GFP-MAPK overexpression. It¡¦s suggested that MAPK is a target protein for SUMOs.

Helicobacter pylori (H. pylori) causes peptic ulcer or gastric cancer through different virulence factors including lipopolysaccharides (LPS), the cytotoxin-associated gene A product (CagA), and vacuolating cytotoxin A (VacA) etc. It stimulated mitogen-activated protein (MAP) kinase signaling cascades. Small ubiquitin-related modifier (SUMO) is a member of ubiquitin-related protein modifiers. However, the mechanisms of the involvement of SUMO-1 on H. pylori induced apoptosis were not clear. Our previous study showed that the expression of RFP-SUMO-1 and apoptosis were increased significantly by fluorescence microscopy assays on RFP-SUMO-1 transfectants during H. pylori infection. In addition, the cytoplasmic SUMO-1 was increased during infection and positively associated with apoptosis. Here, how SUMO-1 was involved in the apoptotic signaling enhancement during H. pylori infection was studied. Results showed that H. pylori infection enhanced MAP kinase activation and the effects were stronger on the SUMO-1 overexpressed cells. However, it was not affected by the secretion of CagA or VacA toxins of H. pylori. To investigate the possible role of SUMO-1 on MAPKs mediated signaling pathways, three selective MAPKs inhibitors were used on RFP-SUMO-1 overexpressed cells. Only p38 inhibitor decreased the levels of apoptosis during H. pylori infection and the expression of p53 was increased on RFP-SUMO-1 1 overexpressed cells. Thus, p38 and p53 pathways were suggested to be involved in SUMO-1 enhanced apoptosis during H. pylori infection. In addition, the nuclear localization of NF-£eB and expression of COX-2 were enhanced on RFP-SUMO-1 overexpressed cells. Moreover, more nuclear NF-£eB and cytoplasmic as well as nuclear RFP-SUMO-1 were observed during H. pylori infection. Our data suggest that H. pylori infection enhances SUMO-1 expression which activates MAPKs on both the pro-apoptotic p38-p53 pathway and the anti-apoptotic ERK-NF-£eB-COX2 pathway. The detail mechanisms on how cells making the final decision on the survival or apoptosis were still not clear and deserving to investigate.

RNF4 belongs to the family of SUMO-targeted ubiquitin E3 ligases (STUbLs). The role of STUbLs in maintaining genomic stability was first discovered in yeast. Theyeast STUbL mutants displayed genomic instability, elevated mutation rates, sensitivity to DNA damaging agents and also demonstrated synthetic lethality with other DNA repair genes. Although the role of vertebrate RNF4 in the DNA damage response was not yet established, it could rescue the Schizosaccaromyces pombe STUbL mutant phenotypes, showing that RNF4 is a functional homologue of the yeast STUbL proteins,and that it might be implicated in the vertebrate DNA damage response.A homozygous knockout of RNF4 in the DT40 chicken lymphocyte cell line was generated to investigate the involvement of vertebrate RNF4 in protecting cells against DNA damage. Although the complete loss of RNF4 did not affect cell proliferation or cell cycle distribution, the RNF4 -/- cells exhibited a selective hypersensitivity to some S-phase specific DNA damaging agents. This hypersensitivity could be rescued by introducing an ortholog of RNF4 from another vertebrate species, and this was dependent on a functional ubiquitin E3 ligase activity of RNF4.To explore the physiological function of RNF4 in the context of a wholeorganism, Danio rerio was chosen as an in vivo model. Danio rerio RNF4 sharedsimilar in vitro biochemical characteristics as RNF4 from other vertebrates – it was able to autoubiquitylate itself and also ubiquitylate SUMO2 chains. In Danio rerio, RNF4 is a maternally provided gene and is highly expressed in the adult gonads. In the ovaries, RNF4 expression was restricted to the early stage oocytes, suggesting a possible role in oocyte development. Loss-of-function studies in Danio rerio were performed using morpholino knockdown and zinc-finger knockout technologies, and the depletion of RNF4 in zebrafish did not affect early embryonic development or viability of the animal.The results presented in this thesis suggests that while vertebrate RNF4 is notlikely to be an essential gene in some vertebrates, it plays a role in the DNA damage response and might be implicated in gonad development in Danio rerio. The zinc-finger knockout model has just been established and a more in-depth analysis is necessary to shed more light on the in vivo functions of RNF4.

No description available.

Nuclear protein KLIP1 cooperates with myeloid leukemia factor 1 (MLF1) to inhibit the programmed cell death resulting in tumor formation. It also inhibits the activity of thymidine kinase promoter of Kaposi¡¦s sarcoma-associated Herpes Virus. KLIP1 functions as a centromere protein, hence acquires its name as CENP-U or CENP-50, to regulate the separation of sister-chromatids during mitosis. These results indicate that KLIP1 plays important roles in regulation of transcription and cell cycle. In this study, six potential SUMO modification sites, K33, K63, K126, K127, K185 and K210, were identified bioinformatically using SUMOplot. Many reports address that SUMO modification alters the transcriptional activity, protein-protein interaction, the subcellular localization and stability of its target protein. Recent data suggest that SUMO is required for centromere binding protein to mediate proper mitotic spindle attachment to the kinetochore, and previous research suggest that there has a SUMO-interaction motif (SIM) in KLIP1 protein sequence. To reveal the interaction between KLIP1 and SUMO-1, and study its effects on KLIP1 function, we co-express GFP-KLIP1 and His-tagged SUMO-1 in HEK 293 cells. After affinity purification of SUMOylated proteins from transfected cells using nickel conjugated beads and subsequent western blotted with anti-GFP. The results indicated the interaction between KLIP1 and SUMO-1 in co-transfected cells. Our confocal microscopy imaging also found colocalization of GFP-KLIP1 with RFP-SUMO-1 nuclear foci. In addition, we failed to detect the interaction between SUMO-1 and mutant KLIP1-M6 ,whose six potential SUMO modified lysine residues were mutated to arginine. Furthermore, we found a distinct nuclear localization of GFP-KLIP1-M6 as compared to the image of wildtype GFP-KLIP1, which show a significant higher frequency of colocalization with RFP-SUMO-1 foci. Taken together, our data suggest the interaction between KLIP1 and SUMO-1 may be related to these six potential lysine residues, which upon mutation blocks its colocalization with SUMO-1 in nuclear foci. The biological significance of their interaction are awaits for further investigation.

For more than fifty years glucocorticoids (GCs) has been used to treat a wide range of inflammatory diseases, such as allergies, asthma, rheumatoid arthritis, and autoimmune diseases, due to its potentiality on the antiinflammatory and immunomodulatory effects. The anti-inflammation actions of glucocorticoid were mediated by an intracellular receptor, glucocorticoid receptor (GR), a member of the nuclear receptor family of ligand-dependent transcription factor. Upon activation by their ligand, GRs translocated to the nuclear and then bound to glucocorticoid responsive element (GRE) or negative glucocorticoid responsive elemen (nGRE). The administration of GCs depended on the acuity of disease and on the responses of patient clinically. Although some Systemic Lupus Erythematosus (SLE) patients given the maximal steroid doses, the response to the therapy remained poorly, and thus called ¡§glucocorticoid resistance¡¨. Despite the fact that the side effects and complications in SLE patients may result from the restrictions of physic; it has been documented that there were some relationships between the glucocorticoid resistance with the polymorphisms of GR, and the levels of glucocorticoid receptor beta. However, no significant differences in the GR polymorphisns (TthIII, ER22/23EK, N363S, BclI and I559N) between controls and SLE patients were found and there were no significant differences found on the levels of SUMO-2 antibody between patients with active and inactive SLE in this study. Neverthless, a significant association on the the allelic polymorphism of BclI was observed in patients with glucocorticoid resistance. Additionally, the expression of GR£] in patients with SLE was higher than that of controls and the TthIII CT genotype was associated with GR£\ expression.

Proteine können post-translational durch Ubiquitin und Ubiquitin-ähnliche Proteine modifiziert werden. Dies erfordert die Aktivität dreier Enzyme. Das Protein wird durch ein E1 Enzym aktiviert, an ein E2 Enzym übertragen und im letzten Schritt mit der Hilfe von E3 Ligasen kovalent an das Substrat gebunden. Dieser Prozess der posttranslationalen Modifikation ist reversibel durch Isopeptidasen. Zu der Proteinfamilie gehören unter anderem Sumo und Nedd8. Beide Proteine sind im Modellorganismus Aspergillus nidulans konserviert. Während die Deletion des nedd8 Homologs neddH zum Zelltod führt, können Pilze ohne SumO überleben. Diese Stämme weisen jedoch Defekte in der sexuellen und asexuellen Entwicklung auf. In dieser Arbeit wurde die NeddH E3 Ligase DcnA/RbxA untersucht. DcnA interagierte mit der Neddylierungs-Maschinerie und die Deletion des Gens führte zu einer leichten Reduktion der Neddylierung von Cullinen. Diese verminderte Neddylierung hatte jedoch keine Auswirkungen auf die pilzliche Entwicklung unter Laborbedingungen. Das RING-finger Protein RbxA zeigt eine E3 Ligaseaktivität sowohl in der Ubiquitinierung als auch in der Neddylierung. Eine Deletion des betreffenden Gens führte zum Zelltod. In einer vorangegangenen Studie mit einem Stamm mit Defekt in der Isopeptidase CSN wurden Substratadaptoren des SCF Ubiquitin-E3-Ligase Komplexes (Fbox-Proteine) identifiziert. In dieser Arbeit wurde festgestellt, dass die biochemische Anreicherung von Fbox15 nicht auf eine generelle Stabilisierung des Proteins zurückzuführen ist, sondern wahrscheinlich auf eine Stabilisierung des SCF Komplexes mit Fbox15. Zusätzlich wurde der Prozess der Sumoylierung in A. nidulans untersucht. Unter normalen Wachstumsbedingungen ist nur ein kleiner Anteil der zellulären Proteine sumoyliert. Um diesen zu erhöhen, wurden die zwei SumO Isopeptidasen UlpA und UlpB untersucht. Durch biochemische Experimente im Wildtyp und einem Stamm, welchem die Isopeptidase UlpA fehlt, konnte ein komplexes SumO Netzwerk identifiziert werden. Zu diesem gehören neben den sumoylierenden Enzymen (E1, E2 und E3), Histon modifizierende Enzyme/Enzymkomplexe, andere Transkriptionsregulatoren, Proteine, die eine Rolle in der RNA-Reifung oder Stressantwort spielen, sowie Wechselwirkungen mit den Prozessen der Ubiquitinierung und Neddylierung. Eine wichtige Schnittstelle zwischen Sumoylierung und Histonmodifikation könnte hierbei der COMPASS Komplex sein. Dieser Komplex ist involviert in Histonmethylierung und damit in die Regulation der Transkription. Um ein besseres Verständnis für die Rolle des Komplexes in der Regulation der pilzlichen Entwicklung zu bekommen, wurde die Kerneinheit SetA deletiert. Der resultierende Stamm zeigte Defekte in der frühen sexuellen Entwicklung, im Koloniewachstum und Sekundärmetabolismus. SetA wurde als wichtiger Faktor für die richtige Positionierung der asexuellen Sporenträger identifiziert.

The uneven success of tropical forest product marketing initiatives over the past decade has illuminated our poor understanding of forest peasant livelihood systems. This dissertation explores how , when and why peoples living within biodiverse tropical forests turn to the sale of forest products to meet their needs over time, through a detailed examination of commercial forest extraction by the Tawahka Sumu (pop. 1,000) of the Mosquitia region, eastern Honduras. The study uses a multi-method, multi-scalar approach that incorporates conceptual insights from cultural ecology, agricultural economics, and peasant studies. / A detailed household census (n = 116, or 88% of Tawahka households in 1998) was used to establish patterns of reliance on commercial extraction. As a group, the Tawahka were found to manage a diverse market income portfolio in which commercial extraction contributed some 18% in 1997--98 (US$23/capita). At the household level, however, reliance on the extractive sector varied from 0--93%. Analysis of multi-year income data suggests that households move easily into, and out of, the sector. Statistical analysis indicates that the most important determinants of this sporadic engagement are unanticipated household-level calamities (illness, crop shortfall). / This ex post insurance function of commercial extraction was also demonstrated over longer time scales by a detailed historical analysis of the Mosquitia's dugout canoe trade, which revealed that the sale of dugout canoes has provided local peoples with an important fall-back during periods of economic recession. Discussion highlights the dynamism of peasant livelihoods, in which forest product sale is seen as only one response to householders' changing needs over both the lifecycle of the household and larger economic cycles in the region. / The modern dynamics of the canoe trade appear to have changed little over two centuries, emphasizing the little-recognized continuity within native exchange systems despite market penetration and monetization. During the 1990s, the Tawahka sold half of the approximately 500 canoes they made, mainly to Miskito buyers. The future of canoe commerce is threatened by pressures on the forests of the newly-created Tawahka Asangni Biosphere Reserve, including high internal growth rates, ladino colonization, and agricultural reorganization in the wake of Hurricane Mitch. The implications of the study's findings to conservation and development initiatives in the neotropics are discussed.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.