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Fas ligand (FasL), a member of the TNF protein family, potently induces cell death by activating its matching receptor Fas. Fas-mediated killing plays a critical role in naturally and pathologically occurring cell death, including development and homeostasis of the immune system. In addition to its receptor-interacting and cell death-inducing extracellular domain, FasL has a well-conserved intracellular portion with a proline-rich SH3 domainbinding site probably involved in nonapoptotic functions. We report here that, as with the Fas receptor, a fraction of FasL is constitutively localized in rafts. These dynamic membrane microdomains, enriched in sphingolipids and cholesterol, are important for cell signaling and trafficking processes. We show that FasL is partially localized in rafts and that increased amounts of FasL are found in rafts after efficient FasL/Fas receptor interactions. Raft disorganization after cholesterol oxidase treatment and deletions within the intracellular FasL domain diminish raft partitioning and, most important, lead to decreased FasL killing. We conclude that FasL is recruited into lipid rafts for maximum Fas receptor contact and cell death-inducing potency. These findings raise the possibility that certain pathologic conditions may be treated by altering the cell death-inducing capability of FasL with drugs affecting its raft localization. ( IntroductionThe Fas ligand (FasL, CD95/Apo-1 ligand, CD178, TNFSF6) molecule belongs to the TNF family and potently induces cell death in Fas (CD95/Apo-1/TNFRSF6) receptor-expressing cells. 1 Fas and FasL interact as oligomers, 2 and crosslinking of Fas leads to the recruitment of the adaptor protein FADD and of to the cytoplasmic Fas domain and to the formation of an intracellular receptor complex called death-inducing signaling complex (DISC). 3 Within the DISC, Casp-8 is activated and triggers the downstream caspase cascade, which executes the apoptotic dismantling of the cell.FasL is a type 2 transmembrane protein consisting of a receptor-interacting extracellular domain and a well-conserved 80-amino acid (aa)-long N-terminal cytoplasmic portion. 1,4,5 Cell death initiated by the Fas/FasL system is important for, among others things, homeostasis of the immune system (eg, activationinduced T-cell death 6 ), cytotoxic T cell (CTL)-mediated killing of virally infected or transformed cells, 7 and immune privilege maintenance. [8][9][10][11][12][13][14] The corresponding experimental studies are supported by the pathology of model systems displaying functional inactivation or aberrant activation of Fas/FasL. Mice carrying loss-of-function mutations in the Fas (lpr) or the Fasl (gld) genes develop lymphadenopathy and splenomegaly with a massive accumulation of CD4 Ϫ CD8 Ϫ T cells. 15 In addition, depending on the genetic background, these animals spontaneously acquire various forms of autoimmune disease with high titers of autoantibodies. 16 In humans, mutations in Fas and Fasl are associated with autoimmune lymphoproliferative syndrome (ALPS). [...

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The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL-and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domaincontaining adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL⅐PSTPIP⅐PTP-PEST complexes which may contribute to FasL reverse signaling.Fas ligand (FasL 3 ; CD95/APO-1 ligand, CD178, TNFSF6) is a 281-amino acid (aa)-long type II transmembrane molecule belonging to the large TNF family of proteins that bind to and activate members of the TNF receptor family (1, 2). FasL and its corresponding receptor Fas (CD95/APO-1) both interact as oligomers (3), and the activated Fas receptor complex initiates a proapoptotic death signal in the receptorbearing cell (4, 5).Although mainly known for its death-promoting activity, FasL has also been studied in the context of further nonapoptotic functions (1, 2, 6). Such experiments are motivated by the special structure of this type II transmembrane protein. In addition to its hydrophobic transmembrane domain, which anchors the molecule within the plasma membrane, and to its extracellular ectodomain, responsible for binding to its receptor, the FasL protein possesses an 80-aa-long N-terminal intracellular part that is responsible for the transduction of (extracellular) signals into FasL-bearing cells and/or that may fulfill regulatory functions. Such a "reverse signaling" has been described in mouse T-cells, which display an altered proliferative behavior upon triggering of their FasL surface molecules (7-9). Results obtained with mouse Sertoli cells, in which FasL engagement leads to mitogen-activated protein kinase activation, as measured by an increase in Erk phosphorylation, also imply stimulatory FasL reverse signaling (10).The importance of the intracellular domain for FasL function and regulation is underlined by its high homology among different species (7,11,12). Several signaling motives within the FasL intracellular domain are highly conserved, including a tandem casein kinase I phosphorylation site (aa 17-21 (11)), a phosphorylatable tyrosine at position 7, a proline-rich region (aa 40 -70) with bona...

DNA mismatch repair (MMR) is essential for the maintenance of replication fidelity. Its major task is to recognize mismatches as well as insertion/deletion loops of newly synthesized DNA strands. Although different players of human MMR have been identified, the regulation of essential steps of MMR is poorly understood. Because MMR is initiated in the nucleus, nuclear import might be a mechanism to regulate MMR. Nuclear targeting is accomplished by conserved signal sequences called nuclear localization signals (NLS), which represent clusters of positively charged amino acids (aa). hMLH1 contains two clusters of positively charged amino acids, which are candidate NLS sequences (aa 469-472 and 496-499), while hPMS2 contains one (aa 574-580). To study the effect of these clusters on nuclear import, NLS mutants of hMLH1 and hPMS2 were generated and expressed in 293T cells. The subcellular localization of the mutant constructs was monitored by confocal laser microscopy. We demonstrated that missense mutations of two signal sequences, one in hMLH1 and one in hPMS2, lead to impaired nuclear import, which was especially prominent for mutants of the hMLH1 residues K471 and R472; and hPMS2 residues K577 and R578.

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Direkt nach dem Examen startete Wiebke Baum voller Elan in ihre erste Arbeitsstelle. Doch auf das, was dann kam, hatte sie w?hrend ihrer Ausbildung keiner vorbereitet.