Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

Análisis del proceso de friction drilling. Viabilidad de la unión de materiales disimilares en uniones atornilladas sin tuerca, compatibilidad de la unión

[ES]En este trabajo se expone un estudio experimental del proceso de taladrado por fricción, más conocido como Friction Drilling y posterior roscado por laminación, en uniones de chapas de acero y aluminio, muy utilizadas en multitud de sectores, que se caracteriza por la ausencia de tuercas. La base de esta técnica es el calor producido por el rozamiento al entrar en contacto la herramienta rotativa con el material, causando el reblandecimiento del material, la fluencia y la deformación de éste. De este modo, se generará una copa cónica, que se roscará por laminación. En este trabajo se va a estudiar la viabilidad del proceso experimentalmente, obteniendo variables de entrada del proceso óptimas que generen una unión de calidad, atendiendo a diferentes aspectos. Sin embargo, se centra sobre todo en analizar la calidad de la unión en lo que se refiere a la compatibilidad de los materiales. Se estudiará la corrosión galvánica por una parte entre acero y aluminio y, por otra parte, entre acero, aluminio y el material del tornillo. Una vez concluido el trabajo, se espera obtener un proceso de unión de materiales disímiles sin tuerca, ofreciendo una mayor calidad que los procesos implementados actualmente.

Análisis del proceso de Friction Drilling: Análisis de la calidad de la junta atornillada

[ES]En el presente trabajo, se pretende optimizar la unión atornillada de chapas de dos materiales disimilares (acero y aluminio) mediante un proceso no convencional, el taladrado por fricción. Dicho proceso está orientado a la calderería fina, sector en el cual tiene gran número de aplicaciones. Se comenzará con una serie de ensayos iníciales y se procederá a realizar pruebas sistemáticas. Se realizarán mediciones de temperaturas, momentos torsores y fuerzas, y se analizaran las tolerancias dimensionales generadas por el proceso para la elección de los parámetros óptimos. El documento se centrará en analizar de forma teórica el comportamiento mecánico de la unión y de los ensayos de tracción correspondientes. Esto servirá para realizar los futuros ensayos de calidad y posteriormente comparar los resultados con los de las uniones convencionales.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Visibility recovery on images acquired in attenuating media. Application to underwater, fog, and mammographic imaging

136 p.