In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

The regulation of NHE₁ and NHE₃ activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/CaM complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1

On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) phosphorylation by protein kinase Cδ (PKCδ) inhibits mitochondria elimination by lysosomal-like structures following ischemia and reoxygenation-induced injury

Background: How damaged mitochondria are removed by mitophagy is not fully described. Results: Ischemia and reoxygenation (I/R)-induced injury triggers mitochondria association of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mitophagy, and protein kinase Cδ (PKCδ) activation inhibits it. Conclusion: PKCδ-mediated phosphorylation of GAPDH inhibits mitophagy. Significance: GAPDH/PKCδ is a signaling switch, which is activated during ischemic injury to regulate the balance between cell survival by mitophagy and cell death by apoptosis.

Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Funktionale Bedeutung der Protein Kinase C - delta (PKC - delta) in der Reorganisation der zytoskelettären Architektur humaner Keratinozyten

Die Kontaktihibition, d.h. die Zell-Zell-Kontakt-vermittelte Proliferationskontrolle, stellt einen fundamentalen Mechanismus zur Aufrechterhaltung der Homöostase in vitro und in vivo dar. So stellen in der Zellkultur nicht-transformierte Zellen in der Regel ihr Wachstum ein, sobald sie einen einschichtigen Zellrasen gebildet haben. Umgekehrt zeichnen sich transformierte Zellen durch einen Verlust der Kontaktinhibition aus. Sie wachsen nach Erreichen eines konfluenten Zellrasens mehrschichtig weiter, und es kommt zur Ausbildung charakteristischer Foci. In dieser Arbeit konnte nachgewiesen werden, dass die Proteinkinase C - delta eine wichtige Funktion in der Regulation der Zytoarchitektur humaner Keratinozyten besitzt und zugleich über Modulation der Zell-Zelladhäsion, insbesondere über Cadherin und Catenin, Einfluss nimmt.

Structural and functional analysis of the hepatitis C virus non-structural protein 5A

Das Hepatitis C Virus (HCV) ist ein umhülltes RNA Virus aus der Familie der Flaviviridae. Sein Genom kodiert für ein ca. 3000 Aminosäuren langes Polyprotein, welches co- und posttranslational in seine funktionellen Einheiten gespalten wird. Eines dieser viralen Proteine ist NS5A. Es handelt sich hierbei um ein stark phosphoryliertes Protein, das eine amphipatische α-Helix im Amino-Terminus trägt, welche für die Membran-Assoziation von NS5A verantwortlich ist. Welche Rolle die Phosphorylierung für die Funktion des Proteins spielt, bzw. welche Funktion NS5A überhaupt ausübt, ist zur Zeit noch unklar. Beobachtungen lassen Vermutungen über eine Funktion von NS5A bei der Resistenz infizierter Zellen gegenüber Interferon-alpha zu. Weiterhin wird vermutet, das NS5A als Komponente des membranständigen HCV Replikasekomplexes an der RNA Replikation beteiligt ist. Das Ziel dieser Doktorarbeit war es, die Funktion von NS5A für die RNA Replikation zu untersuchen. Zu diesem Zweck wurde eine Serie von Phosphorylierungsstellen-Mutanten generiert, die auf Ihre Replikationsfähigkeit und den Phosphorylierungsstatus hin untersucht wurden. Wir fanden, dass bestimmte Serin-Substitutionen im Zentrum von NS5A zu einer gesteigerten RNA Replikation führten, bei gleichzeitig reduzierter NS5A Hyperphosphorylierung. Weiterhin studierten wir den Einfluß von Mutationen in der Amino-terminalen amphipatischen α-Helix von NS5A auf die RNA-Replikation, sowie Phosphorylierung und subzelluläre Lokalisation des Proteins. Wir fanden, dass geringfügige strukturelle Veränderungen der amphipatischen Helix zu einer veränderten subzellulären Lokalisation von NS5A führten, was mit einer reduzierten oder komplett inhibierten RNA Replikation einherging. Zudem interferierten die strukturellen Veränderungen mit der Hyperphosphorylierung des Proteins, was den Schluß nahe legt, dass die amphipatische Helix eine wichtige strukturelle Komponente des Proteins darstellt, die für die korrekte Faltung und Phosphorylierung des Proteins essentiell ist. Als weitere Aspekte wurden die Trans-Komplementationsfähigkeit der verschiedenen viralen Komponenten des HCV Replikasekomplexes untersucht, sowie zelluläre Interaktionspartner von NS5A identifiziert. Zusammenfassend zeigen die Ergebnisse dieser Doktorarbeit, dass NS5A eine wichtige Rolle bei der RNA-Replikation spielt. Diese Funktion wird wahrscheinlich über den Phosphorylierungszustand des Proteins reguliert.

Modulation der Genexpression und des neuronalen Zelltods durch das Amyloid-Vorläufer-Protein (APP)

Das Amyloid-Vorläufer-Protein (APP) spielt eine zentrale Rolle in der Entstehung und Entwicklung von Morbus Alzheimer. Hierbei ist die proteolytische Prozessierung von APP von entscheidender Bedeutung. Das Verhältnis von neurotoxischen und neuroprotektiven Spaltprodukten, die über den amyloidogenen und nicht-amyloidogenen Weg der APP-Prozessierung gebildeten werden, ist für das Überleben von Neuronen und deren Resistenz gegen zytotoxische Stress-Stimuli von hoher Relevanz. Störungen der Calcium-Homöostase sind ein bekanntes Phänomen bei Morbus Alzheimer. Im ersten Teil der vorliegenden Arbeit wurde die Rolle von überexprimiertem APP in der Regulation des neuronalen Zelltods nach Calcium Freisetzung untersucht. Die Calcium Freisetzung aus dem endoplasmatischen Retikulum wurde durch die Inhibition der sarko- und endoplasmatischen Calcium-ATPasen (SERCA) ausgelöst. Dies führt zur Induktion der sogenannten „unfolded protein response“ (UPR) und zu einer Aktivierung von Effektor-Caspasen. Für APP-überexprimierende PC12 Zellen konnte bereits zuvor eine im Vergleich zur Kontrolle nach der durch Calcium Freisetzung-induzierten Apoptose eine erhöhte intrazelluläre Calcium Konzentration nachgewiesen werden. Über die Messung der Aktivierung von Effektor-Caspasen konnte zudem ein gesteigerter Zelltod in den APP-überexprimierenden Zellen gemessen werden. Zudem konnte gezeigt werden, dass der pro-apoptotische Transkriptionsfaktor CHOP, nicht aber die klassischen UPR-Zielgene spezifisch hochreguliert wurden. Die APP-modulierte gesteigerte Induktion von Apoptose nach Calcium Freisezung konnte durch Komplexierung der intrazellulären Calcium Ionen und durch Knockdown von CHOP im Vergleich zur Kontrolle gänzlich unterdrückt werden. Ferner bewirkte die Inhibition der Speicher-aktivierten Calcium-Kanälen (SOCC) eine signifikante Unterdrückung der beobachteten erhöhten intrazellulären Calcium Konzentration und der gesteigerten Apoptose in den APP-überexprimierenden PC12 Zellen. In diesem Teil der Arbeit konnte eindeutig gezeigt werden, dass APP in der Lage ist den durch Calcium-Freisetzung-induzierten Zelltod zu potenzieren. Diese Modulation durch APP verläuft in einer UPR-unabhängigen Reaktion über die Aktivierung von SOCC’s, einer erhöhten Aufnahme von extrazellulärem Calcium und durch erhöhte Induktion des pro-apoptotischen Transkriptionsfaktors CHOP. Im zweiten Teil dieser Arbeit wurde die sAPPα-vermittelte Neuroprotektion untersucht. Dabei handelt es sich um die N-terminale Ektodomäne von APP, die über die Aktivität der α-Sekretase prozessiert wird und anschließend extrazellulär abgegeben wird. Ziel dieser Versuchsreihe war die neuroprotektive physiologische Funktion von APP im Hinblick auf den Schutz von neuronalen Zellen vor diversen für Morbus Alzheimer relevanten Stress-Stimuli bzw. Apoptose-Stimuli zu untersuchen. Durch die Analyse der Effektor-Caspasen konnte gezeigt werden, dass sAPPα in der Lage ist PC12 Zellen potent vor oxidativem Stress, DNA-Schäden, Hypoxie, proteasomalem Stress und Calcium-Freisetzung zu schützen. Außerdem konnte gezeigt werden, dass sAPPα in der Lage ist den pro-apoptotischen Stress-induzierten JNK/Akt-Signalweg zu inhibieren. Eine Beteiligung des anti-apoptotischen PI3K/Akt-Signalwegs bei der sAPPα-vermittelten Protektion konnte über die Inhibition der PI3-Kinase ebenfalls demonstriert werden, die eine Aufhebung der sAPPα-vermittelten Neuroprotektion bewirkte. Diese Daten zeigen neue molekulare Mechanismen auf, die dem sAPPα-vermittelten Schutz vor pathophysiologisch relevanten Stress-Stimuli in neuronalen Zellen zugrunde liegen. Im letzten Teil der Arbeit wurden verschieden Gruppen von pharmakologischen Substanzen im Hinblick auf ihre neuroprotektive Wirkung untersucht und mit ihren Effekten auf den APP-Metabolismus korreliert. Die Untersuchungen ergaben, dass Galantamin, ein schwacher Acetycholinesterase Inhibitor und allosterisch potenzierender Ligand von nikotinischen Acetylcholin-Rezeptoren in der Lage war, naive, und mit noch höherer Effizienz APP-überexprimierende Zelllinien vor dem Stress-induzierten Zelltod zu schützen. Zudem bewirkte Galantamin in APP-überexprimierenden HEK293 Zellen eine rasche Erhöhung der sAPPα Sekretion, so dass hier von einer Rezeptor-vermittelten Modulation des APP Metabolismus ausgegangen werden kann. Omega-3 Fettsäuren wirken sich positiv auf die Membranfluidität von Zellen aus und es konnte bereits gezeigt werden, dass die Bildung des toxischen Aβ Peptids hierdurch vermindert wird. In Analogie zu Galantamin schützte die Omega-3 Fettsäure Docosahexaensäure (DHA) neuronale Zellen vor dem Stress-induzierten Zelltod, wobei der Schutz in APP-überexprimierenden Zellen besonders effizient war. Diese Daten legen nahe, dass die Aktivierung des antiamyloidogenen Wegs der APP-Prozessierung ein viel versprechender Ansatz für die Entwicklung neuer Therapien gegen Morbus Alzheimer sein könnte.

Valutazione del coinvolgimento dell'oncogene c-myc e dei trasportatori abc nella farmacoresistenza dell'osteosarcoma ad alto grado

L’insorgenza di fenomeni coinvolti nello sviluppo della farmacoresistenza costituisce al momento la principale causa di mancata risposta al trattamento chemioterapico nell’osteosarcoma. Questo è in parte dovuto ad una sovraespressione di diversi trasportatori ABC nelle cellule tumorali che causano un aumento dell’efflusso extracellulare del chemioterapico e pertanto una ridotta risposta al trattamento farmacologico. L'oncogene C-MYC è coinvolto nella resistenza al metothrexate, alla doxorubicina e al cisplatino ed è un fattore prognostico avverso, se sovraespresso al momento della diagnosi, in pazienti affetti da osteosarcoma. C-MYC è in grado di regolare l'espressione di diversi trasportatori ABC, probabilmente coinvolti nella resistenza ai farmaci nell’osteosarcoma, e questo potrebbe spiegare l’impatto prognostico avverso dell’oncogene in questo tumore. L’espressione genica di C-MYC e di 16 trasportatori ABC, regolati da C-MYC e / o responsabili dell'efflusso di diversi chemioterapici, è stata valutata su due diverse casistiche cliniche e su un pannello di linee cellulari di osteosarcoma umano mediante real-time PCR. L'espressione della proteina è stata valutata per i 9 trasportatori ABC risultati più rilevanti.Infine l'efficacia in vitro di un inibitore, specifico per ABCB1 e ABCC1, è stata valutata su linee cellulari di osteosarcoma. ABCB1 e ABCC1 sono i trasportatori più espressi nelle linee cellulari di osteosarcoma. ABCB1 è sovraespresso al momento della diagnosi in circa il 40-45% dei pazienti affetti da osteosarcoma e si conferma essere un fattore prognostico avverso se sovraespresso al momento della diagnosi. Pertanto ABCB1 diventa il bersaglio di elezione per lo sviluppo di strategie terapeutiche alternative, nel trattamento dell’osteosarcoma, atte al superamento della farmacoresistenza. L’inibizione dell'attività di tale trasportatore causa un aumento della sensibilità al trattamento chemioterapico nelle linee cellulari di osteosarcoma farmacoresistenti, indicando questo approccio come una possibile strategia per superare il problema della mancata risposta al trattamento farmacologico nei pazienti con osteosarcoma che sovraesprimono ABCB1.

Cellular localization and mechanism of amyloid precursor protein (APP) homodimer formation in an oxidizing environment

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein, which resembles a cell surface receptor, comprising a large ectodomain, a single spanning transmembrane part and a short C-terminal, cytoplasmic domain. It belongs to a conserved gene family, with over 17 members, including also the two mammalian APP homologues proteins APLP1 and APLP2 („amyloid precursor like proteins“). APP is encoded by 19 exons, of which exons 7, 8, and 15 can be alternatively spliced to produce three major protein isoforms APP770, APP751 and APP695, reflecting the number of amino acids. The neuronal APP695 is the only isoform that lacks a Kunitz Protease Inhibitor (KPI) domain in its extracellular portion whereas the two larger, peripheral APP isoforms, contain the 57-amino-acid KPI insert. rnRecently, research effort has suggested that APP metabolism and function is thought to be influenced by homodimerization and that the oligomerization state of APP could also play a role in the pathology of Alzheimer's disease (AD), by regulating its processing and amyloid beta production. Several independent studies have shown that APP can form homodimers within the cell, driven by motifs present in the extracellular domain, as well as in the juxtamembrane (JM) and transmembrane (TM) regions of the molecule, whereby the exact molecular mechanism and the origin of dimer formation remains elusive. Therefore, we focused in our study on the actual subcellular origin of APP homodimerization within the cell, an underlying mechanism, and a possible impact on dimerization properties of its homologue APLP1. Furthermore, we analyzed homodimerization of various APP isoforms, in particular APP695, APP751 and APP770, which differ in the presence of a Kunitz-type protease inhibitor domain (KPI) in the extracellular region. In order to assess the cellular origin of dimerization under different cellular conditions, we established a mammalian cell culture model-system in CHO-K1 (chinese hamster ovary) cells, stably overexpressing human APP, harboring dilysine based organelle sorting motifs at the very C-terminus [KKAA-Endoplasmic Reticulum (ER); KKFF-Golgi]. In this study we show that APP exists as disulfide-bound, SDS-stable dimers, when it was retained in the ER, unlike when it progressed further to the cis-Golgi, due to the KKFF ER exit determinant. These stable APP complexes were isolated from cells, and analyzed by SDS–polyacrylamide gel electrophoresis under non-reducing conditions, whereas strong denaturing and reducing conditions completely converted those dimers to monomers. Our findings suggested that APP homodimer formation starts early in the secretory pathway and that the unique oxidizing environment of the ER likely promotes intermolecular disulfide bond formation between APP molecules. We particularly visualized APP dimerization employing a variety of biochemical experiments and investigated the origin of its generation by using a Bimolecular Fluorescence Complementation (BiFC) approach with split GFP-APP chimeras. Moreover, using N-terminal deletion constructs, we demonstrate that intermolecular disulfide linkage between cysteine residues, exclusively located in the extracellular E1 domain, represents another mechanism of how an APP sub-fraction can dimerize within the cell. Additionally, mutational studies revealed that cysteines at positions 98 and 105, embedded in the conserved loop region within the E1 domain, are critical for interchain disulfide bond formation. Using a pharmacological treatment approach, we show that once generated in the oxidative environment of the ER, APP dimers remain stably associated during transport, reaching the plasma membrane. In addition, we demonstrate that APP isoforms, encompassing the KPI domain, exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-directed cell aggregation of Drosophila Schneider (S2)-cells was isoform independent, mediating cell-cell contacts. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER, suggesting a similar mechanism for heterodimerization. Therefore, dynamic alterations of APP between monomeric, homodimeric, and possibly heterodimeric status could at least partially explain some of the variety in the physiological functions of APP.rn

Induction of RAGE shedding by activation of G-protein-coupled receptors

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNAmediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimers disease and diabetes-induced inflammation.

RAB3GAP1 und RAB3GAP2 (RAB3 GTPase-activating Protein 1/2) beeinflussen die Autophagie in C. elegans und humanen Zellen

Die Proteinhomöostase wird in der Zelle von drei Stoffwechselwegen reguliert: den molekularen Chaperonen, dem Ubiquitin-Proteasom-System und dem autophagosomalen Abbauweg. Die (Makro)Autophagie verpackt und transportiert zytosolische Komponenten in Autophagosomen zu den Lysosomen, wo sie abgebaut werden. Eine Störung dieses Abbauwegs wirkt auf die Proteostase.rnIn dieser Dissertation wurde C. elegans als Modellorganismus zur Erforschung von Proteinstabilität genutzt. In einer RNAi-vermittelten Proteostase-Analyse von Chromosom I und ausgewählter zusätzlicher Gene wurde ein Wurmstamm, der ein Luc::GFP-Konstrukt im Muskel exprimiert, genutzt. Dieses Reporterprotein aggregiert unter Hitzestressbedingungen und diese Aggregation kann durch Modulatoren der Proteostase beeinflusst werden. Dabei wurden mögliche neue Faktoren der Proteinhomöostase entdeckt. Durch weitere Experimente bei denen die Aggregation von PolyQ35::YFP im AM140-System, der Paralyse-Phänotyp und die Akkumulation Thioflavin S-gefärbter Aggregate von Aβ42 im CL2006-Wurmstamm und die Effekte auf die Autophagie mittels eines GFP::LGG1-Konstrukt analysiert wurden, konnten rbg-1 und rbg-2 als neue Modulatoren der Proteinhomöostase, insbesondere der Autophagie, identifiziert werden.rnIm Säuger bilden beide Orthologe dieser Gene, RAB3GAP1 und RAB3GAP2 den heterodimeren RAB3GAP-Komplex, der bisher nur bekannt war für die Stimulation der Umwandlung der GTP-gebundenen aktiven Form zur GDP-gebundenen inaktiven Form der RAB GTPase RAB3. In Immunoblot-Analysen und mikroskopischen Darstellungen im Säugersystem konnte gezeigt werden, dass die Effekte auf die Proteostase über den autophagosomalen Abbauweg wirken. RAB3GAP1/2 wirken als positive Stimulatoren, wenn die Lipidierung von LC3-I und der autophagische Flux von LC3-II und p62/SQSTM1 betrachtet werden. Diese Effekte werden aber nicht über die RAB GTPase RAB3 vermittelt. Die Proteine FEZ1 und FEZ2 haben einen antagonistischen Effekt auf die Autophagie und wenn alle vier Komponenten RAB3GAP1, RAB3GAP2, FEZ1 und FEZ2 zusammen herunter- oder hochreguliert werden, heben sich diese Effekte auf. In Co-Immunopräzipitationen und proteomischen Analysen konnte keine direkte Interaktion zwischen dem RAB3GAP-Komplex und FEZ1/2 oder zu anderen Autophagie-Genen nachgewiesen werden.rnHier konnte der RAB3GAP-Komplex funktionell mit Proteostase und Autophagie in C. elegans und Säugerzellen assoziiert werden. Dieser Komplex zeigt Einflüsse auf die autophagosomale Biogenese indem sie die Proteostase und die Bildung von (prä)autophagosomalen Strukturen in C. elegans und die Lipidierung von LC3 und damit den autophagischen Flux der Autophagiesubstrate LC3-II und p62/SQSTM1 in Säugerzellen beeinflusst. Darüber hinaus wirkt RAB3GAP der komplexen Autophagie-Unterdrückung durch FEZ1 und FEZ2 entgegen. Somit konnte gezeigt werden, dass RAB3GAP als neuartiger Faktor auf die autophagosomale Biogenese und somit auf die Proteostase wirkt.rn

Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases

Meprin and , zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1 , interleukin 18, or tumor growth factor . Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin and a K(i) of 1.1 × 10(-6) M meprin . This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin and inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin activity was 40 times weaker than that of meprin activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin . Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

Postoperative serum pro-calcitonin and C-reactive protein levels in patients with orthopedic infections

QUESTIONS UNDER STUDY / PRINCIPLES: The value of postoperative pro-calcitonin (PCT) in the follow-up of patients with localised infections in the orthopaedic domain is unknown.