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Värmechockprotein 90 kDa (HSP90) bildar ett anmärkningsvärt komplicerat nätverk med en mängd olika cochaperones. Komplexet av FK506-bindande protein 51 kDa (FKBP51) och HSP90 förmedlar proteinveckning och funktion, främjar tau aggregation vid Alzheimers sjukdom och påverkar stressrelaterade störningar, fetma, typ två-diabetes, etc. I samarbete med den molekylära chaperonen HSP90, FKBP51 har nyligen föreslagits som ett lovande terapeutiskt mål för Alzheimers sjukdom (AD). Således skapades knock-in-musen med punktmutationer i tetratricopeptide repeat (TPR) domänen av FKBP51, vilket gör den oförmögen att interagera med HSP90, för att undersöka de potentiella terapeutiska målen för behandling av dessa sjukdomar. Glukokortikoidreceptorn (GR) fungerade traditionellt som utgångspunkten för de initiala studierna av FKBP51-funktion och mekanism som kan stimuleras av den syntetiska glukokortikoiden dexametason (Dexa). Det primära målet med projektet är att förstå den biologiska betydelsen av FKBP51-HSP90 interaktioner. Det är oklart hur FKBP51-mutation påverkar protein-protein-interaktionen och glukokortikoidsignalering. Här analyserades embryonala fibroblaster (MEF) isolerade från vildtyp och FKBP51 mutant mus med avseende på proteinlokalisering, proteinuttryck och genuttryck. Även om ingen säker skillnad mellan vildtyp och mutantmöss sågs i Dexa-medierad glukokortikoidsignalering, förekommer de posttranslationella modifieringarna (PTM) vid exponering för Dexa-behandling av FKBP51 i vildtypmöss i en signifikant högre utsträckning än i Fkbp51mute-möss.Fosforyleringsmodifieringen av FKBP51 antogs initialt och bekräftades av fosforyleringsanrikningsstrategier. Bekräftelse har dock ännu inte erhållits. / Heat shock protein 90 kDa (HSP90) forms a remarkably complicated network with a variety of cochaperones. The complex of FK506-binding protein 51 kDa (FKBP51) and HSP90 mediates protein folding and function, promoting tau aggregation in Alzheimer's disease and influencing stress-related disorders, obesity, type two diabetes, etc. In collaboration with the molecular chaperone HSP90, FKBP51 has recently been proposed as a promising therapeutic target for Alzheimer's disease (AD). Thus, the knock-in mouse harboring point mutations in the tetratricopeptide repeat (TPR) domain of FKBP51 rendering it unable to interact with HSP90 were created to investigate the potential therapeutic targets for the treatment of these diseases. Glucocorticoid receptor (GR) traditionally served as the starting point for the initial studies of FKBP51 function and mechanism which can be stimulated by the synthetic glucocorticoid, dexamethasone (Dexa). The primary goal of the project is to comprehend the biological significance of FKBP51-HSP90 interactions. It is unclear how FKBP51 mutation affects the protein-protein interaction and glucocorticoid signaling. Here, embryonic fibroblasts (MEFs) isolated from wildtype and FKBP51 mutant mouse were analyzed with respect to protein localization, protein expression, and gene expression. Although no certain difference between wildtype and mutant mice was seen in Dexa-mediated glucocorticoid signaling, the post-translational modifications (PTMs) in exposure to Dexa treatment of FKBP51 occur in wildtype mice to a significantly higher extent than in Fkbp51mute mice. The phosphorylation modification of FKBP51 was initially hypothesized and confirmed by phosphorylation enrichment strategies. However, confirmation has not yet been obtained.

Abstract: Neurodegenerative diseases are associated with progressive loss of structure or function of neurons which results in cell death. Recent evidence indicate that all neurodegenerative disorders, sporadic or transmissible, may have a common pathological mechanism at the molecular level. This common feature consists of protein aggregation and accumulation of harmful aggregates in neuronal cells resulting in cellular apoptosis and neurotoxicity. Neurodegenerative diseases can affect abstract thinking, skilled movements, emotional feelings, cognition, memory and other abilities. This diverse group of diseases includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases or transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis. In my project I worked on the molecular mechanism of protein aggregation, propagation and neurotoxicity in Parkinson's disease and prion disease. Prion disease and PD are associated with misfolding and aggregation of PrPc and a-Synuclein (a-Syn), respectively. Despite being two important neurodegenerative disorders, molecular mechanisms of a-Syn or PrPC aggregation and amyloidogenesis are still unclear in PD and prion disease. Furthermore, the toxic protein species in PD have not been characterized yet. In this study we characterize the mechanism of a-Syn and PrPc misfolding in a physiological-like cell free condition in the absence of a-Syn aggregates, PrPc ggregated isoform (Pre's), denaturants or acidic environment. A number of studies indicate that dimerization of PrPc or a-Syn may be a key step in the aggregation process. To test this hypothesis we verified if enforced dimerization of PrPc or a-Syn may induce a conformational change reminiscent of the conversion of PrPc or a-Syn to PrPR' or a-Syn aggregates, respectively. We used a well-described inducible dimerization strategy where a dimerizing domain called FK506-binding protein (Fv) was fused to PrPc or a-Syn in order to produce chimeric proteins Fv-PrP and a-SynF'''. A divalent ligand AP20187 was used to induce protein dimerization. Addition of AP20187 to recombinant Fv-PrP in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet (13-Sheet) structure and simultaneous aggregation of the proteins. However, non-dimerized PrP formed 13-Sheet conformation in very slower rates. In the presence of AP20187, we also report a rapid random coil into 13-sheet conformational transformation of a-SynF" within 24 h, whereas wild type a-Syn showed 24 h delay to achieve P-sheet structure after 48 h. Electron microscopy experiments demonstrated that dimerization induced amyloid fibril formation after 48 h for both Fv-PrP and a-Syr?", whereas in the absence of dimerizing ligand AP20187, PrP or a-Syn converted into amyloid fibrils after 3 days or even later. Dimerization-induced Fv-PrP aggregates were partially resistant to PK digestion which is a characteristics of the naturally occurring PrPR'. The rates of amyloidogenesis in the presence of dimerization was also characterized by Thioflavin T (ThT) fluorescence probing. Whereas the stable structure of Fv-PrP showed no ThT binding for over 60 h of incubation at 37°C, the addition of AP20187 to Fv-PrP resulted in a time-dependent increase in ThT binding. As for a-SynR, dimerization accelerated the rate of ThT binding and amyloid formation comparing to the slower amyloidogenesis rate of wild type a-Syn in the absence of dimerizer AP20187. The impact of dimerization on a-Syn aggregation was further determined by Fluorescence ANS probing, indicating a higher affinity of dimerization-induced a-SynF" aggregates for binding to ANS comparing to wild type a-Syn aggregates. These results indicate that dimerization increases the aggregation and amyloidogenesis processes for Fv-PrP and a-SynF". Both Fv-PrP and a-SynF" amyloids were successfully propagated in vitro by protein misfolding amplification (PMCA) cycle. These results ar in agreement with the theory that all protein aggregates in neurodegenerative diseases propagate with the same molecular mechanism. Neurotoxicity of recombinant Fv-PrP and a-SynF" aggregates was determined in cellulo and in vivo, respectively. Aggregates of Fv-PrP were toxic to cultured cells whilst soluble Fv-PrP and amyloid fibres were harmless to the cells. When injected to the mice brain, both a-Syni" and a-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity in the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by PMCA method, providing the first evidence for the in vitro propagation of synthetic a-Syn aggregates. These results suggest an important role for protein dimerization in aggregation and amyloidogenesis, and therefore, in the pathology of PD and prion disease. The similarities between aggregation, amyloidogenesis and toxicity of PrPC and ct-Syn provide further evidence on the existance of a prion-like mechanism in all neurodegenerative disorders. // Résumé: Les maladies neurodégénératives sont associées à la perte progressive des propriétés structurales ou fonctionnelles des neurones, ce qui engendre la mort des cellules. De récentes études indiquent que tous les désordres neurodégénératifs, sporadiques ou transmissibles, peuvent avoir un mécanisme pathologique commun au niveau moléculaire. Ce dispositif commun se compose de l'agrégation de protéines, de la propagation des agrégats, et de l'accumulation d’agrégats toxiques dans les cellules neuronales, menant à l'apoptose et à la neurotoxicité cellulaire. Les maladies neurodégénératives peuvent affecter la pensée abstraite, les mouvements habiles, les sentiments émotifs, la connaissance, la Mémoire et d'autres capacités cognitives. Ce groupe divers de maladies inclut la maladie d'Alzheimer (AD), de Parkinson (PD), de Huntington (HD), les maladies à prions ou encéphalopathies spongiformes transmissibles (TSEs) et la sclérose latérale amyotrophique (ALS). [symboles non conformes]

No description available.

El inmunosupresor FK506 (Tacrolimus, Prograf) ha incrementado la tasa de supervivencia del trasplante de órganos. FK506 ejerce su acción inmunosupresora mediante la inhibición de la fosfatasa calcineurina en células T activadas. Desgraciadamente, la terapia con FK506 está asociada con efectos no terapéuticos indeseados, entre los que destaca la diabetes, que implican otras dianas distintas de calcineurina. Para identificar estas dianas hemos estudiado la toxicidad celular de FK506 en la levadura de gemación Saccharomyces cerevisiae. FK506 aumentó la sensibilidad de la levadura a estrés osmótico de un modo independiente de calcineurina y las proteínas de unión a FK506. FK506 también indujo un fuerte ayuno de aminoácidos y la activación de la ruta de control general de nutrientes (GCN). La prototrofía de triptófano o el exceso de triptófano eliminó la toxicidad de FK506, lo que muestra que el ayuno de triptófano media este efecto. La mutación de los genes GCN3 y 4 alivió parcialmente la toxicidad de FK506, lo que sugiere que la activación de la ruta GCN por FK506 también está implicada en la tolerancia osmótica. FK506 reforzó la fosforilación de la kinasa Hog1p dependiente de estrés osmótico pero sin inducción de un reportero dependiente de Hog1p. Interesantemente, la interrupción del gen GCN2 suprimió la hiperfosforilación de Hog1p dependiente de FK506 y restauró la actividad del reportero dependiente de Hog1p. A la inversa, la interrupción del gen HOG1 afectó a la activación de Gcn2p y traducción de un reportero GCN4-lacZ dependientes de FK506. Esto pone de manifiesto la existencia de una interacción funcional entre las kinasas Gcn2p y Hog1p. En conjunto estos datos demuestran que tanto el ayuno de aminoácidos como la activación de la ruta GCN inducidos por FK506 contribuyen a la sensibilidad celular a estrés osmótico y revelan un bucle regulador positivo entre las rutas GCN y HOG. Dada la naturaleza conservada de estas rutas, este mecanismo de toxicidad de FK506 / Rodríguez Hernández, CJ. (2006). Identificación de nuevos mecanismos moleculares del inmunosupresor FK506 en Saccharomyces cervisiae [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1849